Full-length genomic RNA of Japanese encephalitis virus, infectious JEV cDNA therefrom, and use thereof

ABSTRACT

The present invention relates to a novel genomic RNA of Japanese encephalitis virus (JEV) and an infectious JEV cDNA therefrom. Particularly, the present invention relates to a full-length genomic RNA of JEV represented by SEQ. ID. No 15 and an infectious JEV cDNA therefrom. JEV genomic RNA and infectious JEV cDNA of the present invention can be used not only for the identification of the JEV genes, but also for the molecular biological studies including JEV replication, transcription, and translation. Moreover, they can also be applied to the development of the therapeutic agents, vaccines, diagnostic reagents, and diagnostic devices for Japanese encephalitis, and can be used as an expression vector for the various foreign genes.

FIELD OF THE INVENTION

The present invention relates to the determination of an authenticJapanese encephalitis virus (JEV) genome RNA sequences, to constructionof infectious JEV cDNA clones, and to utility of the clones or theirderivatives for the purpose of therapeutic, vaccine, and diagnosticapplications. In addition, the invention is also related to JEV vectors,e.g., for heterologous gene expression systems, genetic immunization,and transient gene therapy.

BACKGROUND

JEV is a member of the Flaviviridae family and is transmitted bymosquitoes. It is an important human pathogen that causes permanentneuropsychiatric sequelae and even fatal disease, especially in children(Tsai, Vaccine, 2000, 18(Suppl 2), 1-25; Solomon, NeurologicalInfections and Epidemiology, 1997, 2, 191-199; Umenai et al., Bull.W.H.O., 1985, 63, 625-631). Up to 50,000 cases with a mortality rate ofabout 25% are reported annually, and about half of the survivors exhibitpermanent neuropsychiatric sequelae (Vaughn and Hoke, Epidemiol. Rev.,1992, 14, 197-221; Burke and Leake, Japanese encephalitis, 1988, 63-92,CRC Press Publisher). JEV is distributed mostly in Asia from the formerSoviet Union to India. In recent years, however, transmission of thevirus has recently been observed in the southern hemisphere, indicatingthat this virus could become a worldwide public health threat (Hanna, etal., Med. J. Aust., 1999, 170, 533-536; Hanna, et al., Med. J. Aust.,1996, 165, 256-260; Mackenzie et al., Arch. Virol., 1994, 136, 447-467).

JEV is a small-enveloped virus with a single-stranded, positive-senseRNA genome approximately 11 kb in length. The genome contains a singlelong open reading frame (ORF) flanked by 5′ and 3′ nontranslated regions(NTRs) that are important cis-acting elements for viral replication. TheRNA genome of JEV has a type I cap structure at its 5′-terminus butlacks a poly(A) tail at its 3′ terminus. The ORF is translated into alarge polyprotein that is co- or posttranslationally processed intothree structural and seven nonstructural proteins whose genes arearranged in the genome as follows:C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5 (Lindenbach and Rice,Flaviviridae: The viruses and their replication, 2001, 991-1041,Lippincott Williams&Wilkins Publishers; Venugopal and Gould, Vaccine,1994, 12, 966-975; Chamber et al., Ann. Rev. Microbiol., 1990, 44,649-688). Further information, for example, on the function of themajority of the JEV gene products and the molecular mechanisms involvedin JEV replication, neurovirulence, and pathogenesis, is limited largelybecause of the lack of a reliable reverse genetics system.

Research investigating positive-sense RNA viruses has been considerablyadvanced by the development of the reverse genetics system. Here,infectious cDNA clones of the viral genome in question are constructedand become the templates for infectious RNA synthesis that generatessynthetic viruses. There are two approaches, RNA-launched approach andDNA-launched approach, for the reverse genetics system. In the classical“RNA-launched” approach, cells are transfected with RNA transcripts madefrom the infectious cDNA clones, and the synthetic viruses are thenrecovered from these cells (Satyanarayana et al., Proc. Natl. Acad. Sci.USA, 1999, 96, 7433-7438; van Dinten et al., Proc. Natl. Acad. Sci. USA,1997, 94, 991-996; Liljestrom and Garoff, Biotechnology, 1991, 9,1356-1361; Rice et al., New Biol., 1989, 1, 285-296, Rice et al., J.Virol., 1987, 61, 3809-3819). In an alternative “DNA-launched” approach,synthetic viruses are generated by directly transfecting infectious cDNAclones into susceptible cells. This approach was first reported forpoliovirus (Racaniello and Baltimore, Science, 1981, 214, 916-919), andhas been adapted for alphaviruses (Schlesinger and Dubensky, Curr. Opin.Biotechnol., 1999, 10, 434-439).

Both of these approaches have been used to construct infectious cDNAclones for many positive-sense RNA virus families, includingcoronaviruses, which have the largest RNA genomes (Almazan et al., Proc.Natl. Acad. Sci. USA, 2000, 97, 5516-5521). These clones have beeninvaluable in addressing many questions regarding the positive-sense RNAviruses. However, the construction of a full-length infectious cDNAclone for JEV has been hampered, largely because of the geneticinstability of the cloned cDNA. Despite extensive efforts, a geneticallystable full-length infectious cDNA molecular clone for JEV does notexist (Mishin et al., Virus Res., 2001, 81, 113-123; Zhang et al., J.Virol. Methods, 2001, 96, 171-182; Sumiyoshi et al., J. Infect. Dis.,1995, 171, 1144-1151; Sumiyoshi et al., J. Virol., 1992, 66, 5425-5431).

Thus, the present inventors have disclosed the complete full-lengthnucleotide sequence of the JEV strain CNU/LP2, isolated from a pool ofcirculating mosquitoes in Korea. Based on this sequence, the presentinventors also have developed a convenient and reliable reverse geneticssystem for JEV by synthesizing full-length infectious JEV cDNA molecularclones. The reverse genetics system based on the novel infectious JEVcDNA of the present invention can be effectively used for investigatingthe functions of JEV gene products and other molecular biologicalmechanisms related to replication, neurovirulence, and pathogenesis ofJEV. Further, the present inventors have completed the present inventionby confirming that the infectious JEV cDNA can be effectively used as avector for the heterologous gene expression in a variety of ways.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

It is an object of the present invention to provide an authentic JEVgenome RNA sequences, infectious JEV cDNA clones therefrom, and utilityof the clones or their derivatives for novel gene expression vectors.

To accomplish the above object,

1) The present invention provides an authentic JEV genome RNA sequences.

2) The present invention provides infectious JEV cDNA clones that areable to produce self-replicable JEV RNA transcripts.

3) The present invention provides a JEV-based vector.

4) The present invention provides a self-replicable RNA transcriptsynthesized from the above JEV-based vector.

5) The present invention provides a recombinant JEV virus obtained fromcells transfected with a synthetic RNA transcript synthesized from theJEV-based vector.

6) The present invention provides a JEV-based expression vector.

7) The present invention provides a variety of strategies for expressingheterologous genes using the JEV-based expression vector.

Further features of the present invention will appear hereinafter.

I. The present invention provides an authentic JEV genome RNA sequences.

Korean isolate JEV genomic RNA of the present invention is composed of a5′nontranslated region (NTR), a polypeptide coding region and a 3′NTR.Particularly, the full-length RNA genome is 10,968 bp in length andconsists of a 95 bp 5′NTR followed by a 10,299 bp single open readingframe and terminated by a 574 bp 3′NTR.

According to the preferred embodiment of the present invention, thenovel genomic RNA of JEV has a sequence represented by SEQ. ID. No 15.And the novel genomic RNA of the present invention also includes anysequence having 98% homology with JEV genomic RNA represented by SEQ.ID. No 15.

Korean isolate JEV of the present invention was isolated and purifiedfrom Korean JEV strain K87P39 by taking advantage of plaque-purificationtechnique, and was named “JEV CNU/LP2” (see FIG. 1).

In order to determine the complete nucleotide sequence of CNU/LP2, aKorean isolate JEV, the present inventors amplified the entire viral RNAgenome apart from the 5′ and 3′ termini using long reversetranscription-polymerase chain reaction (RT-PCR) and yielded threeoverlapping cDNA products denoted JVF (nucleotide (nt) 1-3865), JVM (nt3266-8170), and JVR (nt 7565-10893) (about 3.9, 4.9, and 3.3 kb inlength, respectively) (see FIG. 2A).

The 3′-terminal sequence of CNU/LP2 viral RNA was analyzed aftersynthetic oligonucleotide T was ligated to it. Oligonucleotide T servesas a specific priming site for cDNA synthesis and PCR amplification (seeFIG. 2B). Agarose gel electrophoresis revealed that the amplifiedproducts migrated as two bands, a larger band of approximately 700 bpand a smaller band of about 450 bp (see FIG. 2C). Both bands werepurified and cloned, and 20 and 10 randomly picked clones containing thelarger and the smaller bands, respectively, were sequenced. As has beendocumented for most of the fully sequenced JEV isolates, the presentinventors found that all the clones with the larger insert (about 700bp) terminated the viral genome with -GATCT¹⁰⁹⁶⁸. In contrast, all theclones with the smaller insert (about 450 bp) showed the viral genometruncated at nt 10,684, resulting in a band 284 bp shorter. Duringassembly of the full-length JEV cDNA, the present inventors used thenucleotide sequences of the larger insert because the smaller insert didnot contain 284 nucleotides at the 3′ end of the viral genome.

The 5′-terminal sequence of CNU/LP2 viral RNA was examined after the capstructure at its 5′ end had been removed by incubation with tobacco acidpyrophosphatase. The resulting viral RNA was then self-ligated, and the3′-5′ junction was subjected to cDNA synthesis and PCR amplificationwith a positive-sense primer for RT-PCR complementary to a sequence nearthe viral 3′ end (nt 10259-nt 10276) and a negative-sense primercorresponding to a sequence near the viral 5′ end (nt 164-nt 181) (seeFIG. 2D). Agarose gel electrophoresis revealed the amplified products asa single band of about 850 bp (see FIG. 2E). The amplicons were cloned,and 12 randomly picked clones were sequenced. In all 12 clones, the-GATCT¹⁰⁹⁶⁸ of the viral 3′-terminal sequence was followed by the5′-terminal sequence ¹AGAAGT- (see FIGS. 2B and 2C).

Thus, the present inventors have determined the complete nucleotidesequence of the JEV CNU/LP2 isolate represented by SEQ. ID. No 15. Thefull-length RNA genome of JEV CNU/LP2 is 10,968 bp in length andconsists of a 95 bp 5′NTR followed by a 10,299 bp single open readingframe and terminated by a 574 bp 3′NTR. The present inventors comparedthe complete nucleotide sequence of the CNU/LP2 isolate with sequencesof all 26 JEV strains (Ishikawa, K94P05, FU, CH2195LA, CH2195SA, RP-2ms, RP-9, CH1392, T1P1, YL, JaGAr01, HVI, TC, TL, Beijing-1, Ling,Vellore P20778, p3, SA14-14-2, SA(A), SA14-12-1-7, SA14-2-8, SA14,SA(V), GP78, and JaOArS982) available in GenBank database. Suchinformations concerning viral strains used for the comparison asisolation regions, isolation years, sources and GenBank accessionnumbers are briefly stated hereinafter (see Table 1).

TABLE 1 GenBank Geographic accession location Year Strain Source numberAustralia 1995 FU Human serum AF217620 China 1954 SA14 Mosquito U14163SA14-14-2 SA14 AF315119 derivative SA14-12-1-7 SA14 AF416457 derivativeSA14-2-8 SA14 U15763 derivative SA(V) SA14 D90194 derivative SA(A)SA14-14-2 D90195 derivative 1949 Beijing-1 Human brain L48961 1949 p3Mosquito U47032 India 1978 GP78 Human brain AF075723 1958 Vellore Humanbrain AF080251 P20778 Japan 1982 JaOArS982 Mosquito M18370 IU IshikawaIU AB051292 1959 JaGAr01 Mosquito AF069076 Korea 1994 K94P05 MosquitoAF045551 1987 CNU/LP2 Mosquito This invention Taiwan 1997 T1P1 MosquitoAF254453 1994 CH2195LA CH2195 AF221499 derivative 1994 CH2195SA CH2195AF221500 derivative 1990 CH1392 Mosquito AF254452 1985 RP-2ms MosquitoAF014160 1985 RP-9 Mosquito AF014161 1965 Ling Human brain L78128 IU YLIU AF486638 IU TC Mosquito AF098736 IU TL Mosquito AF098737 IU HVIMosquito AF098735 IU: Information unavailable

From the comparison of the nucleotide sequence of the CNU/LP2 isolatewith nucleotide sequences of other JEV strain, it was shown that the JEVisolate CNU/LP2 genome shared various degrees of sequence similaritywith these other genomes [89.0% (Ishikawa), 89.1% (K94P05), 89.3% (FU),95.8% (CH2195LA), 95.9% (RP-2 ms), 97.2% (RP-9), 97.3% (CH1392), 97.3%(T1P1), 97.0% (YL), 97.4% (JaGAr01), 97.1% (HVI), 96.9% (TC), 96.7%(TL), 96.4% (Beijing-1), 96.3% (Ling), 96.0% (Vellore P20778), 97.1%(p3), 97.4% (SA14-14-2), 97.5% (SA(A)), 97.5% (SA14-12-1-7), 97.7%(SA14-2-8), 97.9% (SA14), 97.9% (SA(V)), 96.3% (GP78), and 97.1%(JaOArS982)] (see Table 2). Therefore, the nucleotide sequences of JEVviral genomic RNA having over 98% sequence similarity with thenucleotide sequence of the present invention represented by SEQ. ID. NO15 can be included in the category of the claim of the presentinvention.

TABLE 2 % sequence identity^(a) Isolate Ishkawa K94P05 FU CH2195LACH2195SA RP-2m s RP-9 CH1392 T1P1 YL JaGAr01 HVI TC TL Ishkawa 97.0 90.188.3 88.3 88.9 89.0 89.1 89.1 88.9 89.2 89.0 88.9 88.6 K94P05 97.7 89.688.6 88.6 89.2 89.3 89.4 89.4 89.1 89.5 89.2 89.1 88.0 FU 97.7 97.0 88.988.9 89.3 89.4 89.4 89.4 89.2 89.5 89.4 89.2 89.1 CH2195LA 97.7 97.089.9 89.9 96.2 96.3 96.3 96.3 86.1 96.5 86.2 95.9 95.8 CH2195SA 97.196.5 99.0 99.0 96.3 96.3 96.3 96.3 96.1 96.5 96.2 95.9 95.8 RP-2m s 97.596.8 99.4 99.4 98.8 99.9 99.5 99.5 99.3 89.4 98.8 88.2 96.1 RP-9 97.696.9 89.5 99.5 88.9 99.7 99.6 99.5 89.3 98.5 88.8 98.2 96.2 CH1392 97.897.2 99.7 99.7 99.1 99.6 99.7 98.9 99.5 89.7 89.0 98.3 98.3 T1P1 97.596.8 89.3 99.3 98.7 99.1 99.2 99.4 89.5 99.7 89.0 98.3 98.3 YL 97.4 96.799.2 99.2 98.7 89.1 99.2 99.4 89.0 89.4 98.8 88.1 98.0 JaGAr01 97.1 96.498.9 88.9 98.2 98.7 98.8 99.1 96.6 98.7 99.1 88.4 88.4 HVI 97.2 96.598.9 98.9 98.3 98.8 98.9 99.1 98.7 98.8 98.7 98.6 98.4 TC 97.0 96.4 98.898.6 98.2 88.7 98.8 99.0 98.6 98.5 88.2 98.4 89.0 TL 87.2 96.6 89.0 99.088.4 96.8 88.9 99.2 98.8 98.7 96.4 88.5 99.7 Beijing-1 97.3 96.6 99.099.0 98.5 98.9 99.0 89.2 98.8 88.7 96.4 98.6 99.2 99.3 Ling 97.4 96.789.1 99.1 98.5 99.0 99.1 99.3 96.9 88.9 98.6 98.7 99.0 99.2 VelloreP20778 97.7 97.1 99.5 99.5 98.9 99.4 99.5 99.7 99.3 99.2 98.9 99.1 99.199.2 p3 97.8 97.1 99.5 99.5 98.8 99.4 99.5 99.7 99.4 99.3 99.0 99.2 99.099.2 SA14-14-2 97.9 97.1 99.5 99.5 98.9 99.3 99.4 89.7 89.2 99.2 98.989.0 98.8 89.0 SA(A) 97.1 96.6 98.8 98.8 98.2 98.7 98.6 99.0 96.6 98.586.2 98.3 98.1 98.3 SA14-12-1-7 97.2 96.6 88.9 98.8 88.3 96.7 98.9 99.198.7 88.6 98.3 98.4 96.2 98.4 SA14-2-8 97.7 97.3 99.4 99.4 98.8 99.399.4 99.6 99.2 99.2 98.8 98.9 98.8 98.9 SA14 97.5 96.7 89.0 99.0 98.496.9 89.0 99.2 98.9 96.6 98.6 98.7 98.4 98.5 SA(V) 97.3 96.6 98.9 98.998.3 98.7 98.8 99.1 98.7 96.6 98.4 98.5 98.2 98.4 CNU/LP2 97.4 96.7 96.989.9 96.2 98.7 98.8 99.1 88.6 83.6 88.2 88.4 88.2 88.4 GP78 97.0 96.498.6 88.6 98.0 96.5 98.6 98.8 98.5 98.4 88.0 98.1 98.0 88.2 JaOArS98297.6 96.6 97.8 97.6 97.2 97.7 97.8 98.0 97.6 97.6 97.2 97.3 97.1 97.3WNV 76.2 75.8 76.6 76.6 76.1 76.4 76.5 76.7 76.5 76.5 76.3 76.4 76.176.2 % sequence identity^(a) Vellore SA14- SA14- Isolate Beijing-1 LingP20778 p3 14-2 SA(A) 12-1-7 SA14-2-8 SA14 SA(V) CNU/LP2 GP78 JaOArS982WNV Ishkawa 88.9 88.8 88.8 89.1 88.8 88.9 88.9 89.1 89.2 89.2 89.0 68.868.9 69.0 K94P05 89.0 89.0 68.9 89.4 89.1 89.2 69.2 89.3 89.5 89.5 89.169.0 68.5 68.7 FU 69.3 89.0 89.1 89.4 89.1 89.2 89.2 89.3 69.6 89.6 89.368.7 69.4 69.5 CH2195LA 95.6 95.7 95.1 96.3 96.6 96.7 96.7 96.8 97.197.1 85.8 95.7 97.3 69.4 CH2195SA 95.6 95.7 95.1 96.3 96.6 96.7 96.796.9 97.1 97.1 95.9 95.7 97.3 69.5 RP-2m s 97.2 97.1 96.6 97.9 98.0 98.198.1 98.3 98.5 98.5 97.1 98.9 97.7 69.4 RP-9 97.2 97.2 96.7 98.0 98.198.1 98.1 98.3 98.5 98.5 97.2 96.9 97.6 69.4 CH1392 97.3 97.3 96.8 98.298.2 98.3 98.3 98.4 86.6 98.7 97.3 97.0 97.8 69.4 T1P1 97.3 97.3 96.898.1 88.2 98.2 88.3 98.4 98.6 98.6 97.3 97.0 97.8 69.4 YL 97.1 97.1 96.597.9 98.0 98.0 98.3 98.2 98.4 98.4 97.0 96.8 97.6 69.2 JaGAr01 97.4 97.496.9 98.3 96.3 98.4 98.4 98.5 98.8 98.8 97.4 97.1 98.0 69.5 HVI 97.297.2 96.7 98.1 96.1 99.1 98.1 98.3 98.6 98.5 97.1 96.9 97.7 69.4 TC 97.297.1 96.4 97.7 97.8 97.9 97.8 98.1 98.3 93.3 86.9 96.7 97.4 69.5 TL 97.096.8 86.1 97.5 97.8 97.7 97.7 97.9 98.1 98.1 96.7 96.5 97.2 69.3Beijing-1 99.1 96.7 97.4 97.2 97.2 97.3 97.5 97.6 97.6 86.4 98.1 97.069.5 Ling 99.2 96.7 97.3 97.1 97.2 97.2 97.4 97.6 97.6 86.3 95.2 97.269.5 Vellore P20778 99.3 99.4 96.8 96.8 96.7 96.7 96.9 97.1 97.1 88.095.6 96.4 69.5 p3 99.2 99.5 99.6 96.2 98.3 98.3 98.5 98.7 98.7 97.1 97.197.8 69.5 SA14-14-2 89.1 99.2 99.6 89.6 89.8 99.6 99.4 89.4 99.4 97.497.3 88.1 69.4 SA(A) 88.4 96.5 98.8 88.9 96.9 99.6 99.4 99.4 99.4 97.597.4 88.2 69.4 SA14-12-1-7 98.5 93.6 99.0 99.0 96.9 99.7 99.4 99.5 89.597.5 87.4 96.2 69.4 SA14-2-8 99.0 99.2 99.5 99.6 99.5 89.1 99.2 99.689.6 97.7 97.6 96.3 69.5 SA14 98.6 98.7 89.1 99.2 99.2 99.5 98.5 99.289.9 97.9 97.6 98.6 69.6 SA(V) 98.5 98.6 98.9 89.0 99.0 98.3 93.4 99.099.5 97.9 97.8 98.6 69.6 CNU/LP2 88.5 88.6 98.9 89.0 89.0 98.4 98.4 89.098.6 88.5 98.3 97.1 59.5 GP78 98.3 98.3 98.7 88.7 98.7 98.1 88.2 98.788.2 98.2 88.2 87.2 69.6 JaOArS982 97.4 97.5 97.9 97.9 98.0 97.3 97.497.9 97.6 97.4 97.5 97.1 69.6 WNV 76.3 76.4 76.6 76.6 76.8 76.2 76.376.7 76.6 76.5 76.4 76.5 76.7 ^(a)The percent nucleotide sequenceidentities of the complete genomes are presented at the upper right. Thepercent amino acid sequence identities of the complete genomes are shownin the lower left. The percentages of CNU/LP2 sequence identities areindicated in boldface type.

In addition to determine the nucleotide sequence of polypeptide codingregion of JEV, the nucleotide sequences of 5′ and 3′NTRs includingcis-acting elements involved in the regulation of viral replication,transcription, and translation of the virus were also determined bytaking advantage of molecular biological approaches. The importance ofboth regions have been supported by some of earlier studies reportingthat both the 5′- and 3′-terminal regions are required for theinitiation of flavivirus RNA replication in vitro (You and Padmanabhan,J. Biol. Chem., 1999, 274, 33714-33722) and in vivo (Khromykh et al., J.Virol., 2001, 75, 6719-6728). Especially, ¹AGAAGT- and -GATCT¹⁰⁹⁶⁸,which were proved to be the nucleotide sequence of 5′- and 3′-terminalregions of JEV CNU/LP2 in the present invention, are highly expected toplay an important role in self-replication of the virus.

The present inventors proved through the experiments illustratedhereinafter that infectious synthetic JEV could be produced when cellswere transfected with a synthetic RNA transcript having a full-lengthnucleotide sequence of JEV, and further, the inventors are the first toprove the function of the complete full-length nucleotide sequence whichis necessary for JEV self-replication.

II. The present invention provides infectious JEV cDNA clones that areable to produce a self-replicable JEV RNA transcripts.

The infectious JEV cDNA clones of the present invention was synthesizedwith a nucleotide sequence represented by SEQ. ID. No 15 or nucleotidesequences of full-length JEV genomic RNA having over 98% sequencesimilarity therewith, and was used as a template for the synthesis ofself-replicable JEV RNA transcript through in vitro transcription. Inorder to construct the full-length JEV cDNA clones, a viral genomic RNAincluding 5′- and 3′-terminal regions should be amplified by RT-PCR andthen the obtained overlapping cDNAs were sequentially assembled.

In order to produce a full-length synthetic JEV RNA transcript throughin vitro runoff transcription reaction, SP6 or T7 promoter transcriptionstart site was located at the front of 5′-end of JEV genomic RNA and aunique restriction endonuclease recognition site was located at the endof the viral genome (see FIG. 3A). In the preferred embodiment of thepresent invention, three SP6-driven full-length JEV cDNAs and threeT7-driven full-length JEV cDNAs were constructed by using threeoverlapping JEV cDNAs (JVF, JVM and JVR) and two additional cDNAs; oneis corresponding to 5′-terminal region including SP6 or T7 promotersequence and the other is corresponding to 3′-terminal region includingXho I and Xba I recognition sequence as a runoff site (see FIGS. 3B and3C). However, it is a common knowledge for the people in this field thatother promoters but the above two promoters can be used as well. Thefull-length JEV cDNA developed in the present invention uses Xho I andXba I as a runoff site but other restriction enzymes can be used ascommonly known.

The JEV cDNA clones of the present invention are constructed byproducing subclones containing many overlapping cDNAs using thebacterial artificial chromosome (BAC) plasmid pBeloBAC11 as a vector andsequentially linking those subclones into the full-length JEV cDNAs.

In the preferred embodiment of the present invention, the presentinventors provide one set of three JEV cDNA clones having SP6 promoterand represented by SEQ. ID. No 43, No 44, and No 45, respectively. Inaddition, the present inventors also provide the other set of three JEVcDNA clones having T7 promoter and represented by SEQ. ID. No 46, No 47,and No 48, respectively (see FIGS. 3B and 3C). To ensure that the 3′ endof the viral genome after runoff transcription would be close toauthentic, in all cases, the present inventors placed a uniquerestriction endonuclease recognition site, either Xho I or Xba I, at theend of the viral genome (see FIGS. 3B and 3C).

III. The present invention provides a JEV-based vector.

The vector of the present invention is characterized by including afull-length infectious JEV cDNA. In the preferred embodiment of thepresent invention, the inventors provide vectors‘pBAC.sup.SP6/JVFL/XhoI’, ‘pDBAC.sup.SP6/JVFLx/XhoI’, and‘pBAC.sup.SP6/JVFLx/XbaI’ which all have SP6 promoter and each isrepresented by SEQ. ID. No 43, No 44, and No 45, and also vectors‘pBAC.sup.T7/JVFL/XhoI’, ‘pBAC.sup.T7/JVFLx/XhoI’, and‘pBAC.sup.T7/JVFLx/XbaI’ which all have T7 promoter and each isrepresented by SEQ. ID. No 46, No 47, and No 48.

The present inventors deposited two most efficient vectors of the above,pBAC^(T7)/JVFLx/XbaI and pBAC^(SP6)/JVFLx/XbaI, at Gene Bank of KoreaResearch Institute of Bioscience and Biotechnology (KRIBB) on Oct. 2,2002 (Accession No: KCTC 10346BP, KCTC 10347BP). All restrictionsimposed by the depositor on the availability to the public of thedeposited material will be irrevocably removed upon the granting of apatent.

IV. The present invention provides a self-replicable RNA transcriptsynthesized from the above JEV-based vector.

For in vitro runoff transcription, JEV cDNA templates were linearized bydigestion with Xho I or Xba I which is engineered for run-off site rightbehind 3′-terminal region of the viral genome (see FIG. 3). SP6polymerase runoff transcription of the two Xho I-linearized plasmids(pBAC^(SP6)/JVFL/XhoI and pBAC^(SP6)/JVFLx/XhoI) in the presence of them⁷G(5′)ppp(5′)A cap structure analog yielded capped synthetic RNAscontaining three nucleotides (CGA) of virus-unrelated sequence at their3′ ends (see FIG. 3B). This is the result of copying the 5′ overhangleft by the Xho I digestion. Similarly, SP6 polymerase runofftranscription of the Xba I-linearized pBAC^(SP6)/JVFLx/XbaI plasmid inthe presence of the m⁷G(5′)ppp(5′)A cap structure analog produced cappedsynthetic RNAs with four nucleotides (CTAG) of virus-unrelated sequenceat their 3′ ends (see FIG. 3B).

The present inventors have performed infectious center assay to measurethe specific infectivity of the synthetic JEV RNA transcripts. As aresult, when susceptible BHK-21 cells were transfected with thesynthetic RNA transcripts, all were highly infectious (3.4-4.3×10⁵PFU/μg) (see Table 3). Similar results (2.9-3.8×10⁵ PFU/μg) were alsoobtained with synthetic RNAs transcribed from the T7-driven cDNAconstructs by T7 polymerase runoff transcription (see Table 3).

It has been reported that for some flaviviruses, the presence ofvirus-unrelated sequences at the 3′ end of synthetic RNAs transcribedfrom infectious cDNA diminishes or abrogates their specific infectivity(Yamshchikov et al., Virology, 2001, 281, 294-304). Based on thisreport, the present inventors generated synthetic RNAs lackingvirus-unrelated sequences at their 3′ends and compared their specificinfectivities. Particularly, the present inventors generated syntheticRNAs lacking the unrelated sequences by treating the Xba I-linearizedpBAC^(SP6)/JVFLx/XbaI plasmid with mung bean nuclease (MBN) prior to thetranscription reaction, which removed the four excess nucleotides ofCTAG. To verify MBN activity, Xba I-linearized and MBN-treatedpBAC^(SP6)/JVFLx/XbaI plasmid was self-ligated, and its viral 3′ end wassequenced, demonstrating removal of the four excess nucleotides of CTAG.RNA transcripts from Xba I-linearized and MBN-treatedpBAC^(SP6)/JVFLx/XbaI and pBAC^(T7)/JVFLx/XbaI(pBAC^(SP6)/JVFLx/XbaI^(MBN), see FIG. 3B andpBAC^(T7)/JVFLx/XbaI^(MBN), see FIG. 3C) both had increased specificinfectivities compared to the untreated transcripts. Precisely, thespecific infectivity of RNAs transcribed frompBAC^(SP6)/JVFLx/XbaI^(MBN) was estimated to be 3.1×10⁶ PFU/μg,approximately 10-fold higher than the specific infectivity (3.4×10⁵PFU/μg) of the unmodified template (see Table 3, infectivity). The RNAsderived from pBAC^(T7)/JVFLx/XbaI also had increased specificinfectivity after MBN modification (2.7×10⁶ PFU/μg) (see Table 3,infectivity). Therefore, the present inventors confirmed that theauthentic 3′ end of the JEV genome should be present to ensure highlyinfectious synthetic JEV RNA transcripts are generated. Thus, theinfectious JEV cDNA clones of the present invention could be used astemplates for runoff transcription that generated highly infectioussynthetic RNAs with a specific infectivity of 10⁵ to 10⁶ PFU/μg.

Previous attempts (Mishin et al., Virus Res., 2001, 81, 113-123; Zhanget al., J. Virol. Methods, 2001, 96, 171-182; Sumiyoshi et al., J.Infect. Dis., 1995, 171, 1144-1151; Sumiyoshi et al., J. Virol., 1992,66, 5425-5431) to assemble a full-length infectious JEV cDNA were allfailed because of the genetic instability of cloned JEV cDNA. One studyattempted to overcome this problem by designing a system in which thetemplate would be generated by in vitro ligation of two overlapping JEVcDNAs (Sumiyoshi et al., J. Virol., 1992, 66, 5425-5431). This templatewas then used to synthesize infectious RNA transcripts in vitro.However, the specific infectivity of these transcripts was about 100PFU/μg, which was too low to make this system useful for molecular andgenetic analyses of virus biology (Sumiyoshi et al., J. Virol., 1992,66, 5425-5431).

In the present invention, the present inventors were able to overcomethe genetic instability of JEV cDNA by cloning it into a BAC plasmidthat is maintained at one or two copies in E. coli. The geneticstructure and functional integrity of the infectious cDNA plasmidremained stable for at least 180 generations during its propagation inE. coli (see FIG. 7). So, the present inventors settled the problem ofgenetic instability of making full-length infectious JEV cDNA byintroducing BAC, and further had skills to treat the syntheticinfectious JEV cDNA stably.

It is important to produce full-length infectious JEV cDNA that, in invitro transcription, would generate RNA transcripts with authentic 5′and 3′ ends because several studies have shown that both the 5′- and3′-terminal regions are needed for the initiation of flavivirus RNAreplication in vitro (You and Padmanabhan, J. Biol. Chem., 1999, 274,33714-33722) and in vivo (Khromykh et al., J. Virol., 2001, 75,6719-6728). To achieve this objective, the present inventors adaptedapproaches used previously for other flaviviruses (van der Werf et al.,Proc. Natl. Acad. Sci. USA, 1986, 83, 2330-2334; Rice et al., New Biol.,1989, 1, 285-296). The cap structure in JEV genomic RNA is followed bythe dinucleotide AG, an absolutely conserved feature of the flaviviruses(Rice, Flaviviridae: The viruses and their replication, 1996, 931-960,Lippincott-Raven Publisher). The authenticity of the 5′ end was ensuredby placing either the SP6 or the T7 promoter transcription start at thebeginning of the viral genome. Incorporating the m⁷G(5′)ppp(5′)A capstructure analog in the SP6 or T7 polymerase-driven transcriptionreactions (Contreras et al., Nucleic Acids Res., 1982, 10, 6353-6362),the present inventors synthesized capped RNA transcripts with authentic5′ ends that were highly infectious upon transfection into susceptiblecells. In addition, incorporating the m⁷G(5′)ppp(5′)G cap structureanalog in the SP6 or T7 polymerase-driven transcription reactions(Contreras et al., Nucleic Acids Res., 1982, 10, 6353-6362) places anunrelated extra G nucleotide upstream of the dinucleotide AG. Asreported earlier (Rice et al., New Biol., 1989, 1, 285-296), the presentinventors did find that the extra nucleotide was lost from the genomicRNA of the recovered JEV progeny. Furthermore, the present inventors didnot observe that the infectivity or the replication of synthetic RNAstranscribed from infectious cDNA templates was altered if the inventorsadded the extra nucleotide.

The dinucleotide CT located at the 3′ end of JEV RNA is absolutelyconserved among the flaviviruses (Rice, Flaviviridae: The viruses andtheir replication, 1996, 931-960, Lippincott-Raven Publisher). Thissuggests that these nucleotides are important in viral replication andthat transcripts from infectious cDNAs must have authentic 3′ ends.Thus, the present inventors designed our reverse genetics system for JEVso that the synthetic RNA would be terminated with the authentic 3′ends. Indeed, the present inventors showed that RNA transcripts withauthentic 3′ ends were 10-fold more infectious than transcripts withthree or four virus-unrelated nucleotides hanging on their 3′ ends.

V. The present invention provides a recombinant JEV virus obtained fromcells transfected with a synthetic RNA transcript synthesized from theJEV-based vector.

In the present invention, synthetic JEV viruses produced from the cellstransfected with JEV RNA transcripts synthesized from full-lengthinfectious JEV cDNAs were produced. Transfected cells showed strongcytopathic effect induced by JEV virus infection and all the syntheticviruses were indistinguishable from the CNU/LP2 parental virus in termsof plaque morphology, cytopathogenicity, growth kinetics, proteinexpression and RNA accumulation (see FIG. 5). Furthermore, recombinantJEV virus mutants could be produced by inducing site-directed mutationon a specific region of JEV cDNA, indicating that the infectious JEVcDNA can be manipulated in E. coli. Thus, the reverse genetics systemusing the infectious JEV cDNAs of the present invention can beeffectively used for the genetic studies on the replication mechanism ofJEV genome.

VI. The present invention provides a JEV-based expression vector.

The present invention provides the use of JEV cDNA as a novel expressionvector in a variety of cell types. Alphaviruses, which are also RNAviruses, can replicate in a variety of commonly used animal cells andthus have been successfully exploited as eukaryotic expression vectorsin cell culture and in vivo (Agapov et al., Proc. Natl. Acad. Sci. USA,1998, 95, 12989-12944; Frolov et al., Proc. Natl. Acad. Sci. USA, 1996,93, 11371-11377; Schlesinger, Trends Biotechnol., 1993, 11, 18-22). Itwas reported that JEV, like the alphaviruses, is also able to replicatein a wide variety of primary and continuous cell cultures from humans,mice, monkeys, pigs, and hamsters (Burke and Monath, Flaviviruses, 2001,1043-1125, Lippincott Williams&Wilkins Publishers). This suggests thatJEV could be useful as a vector for the expression of heterologous genesin a variety of different cells. When a full-length infectious JEV cDNAis used as an expression vector, in which heterologous genes areinserted, RNA transcripts having heterologous genes are produced by invitro transcription reaction. Those transcripts can self-replicate asthey are transfected into cells, so that lots of foreign proteins can beproduced.

An expression cassette is preferably inserted at the beginning of JEV3′NTR for the expression of a heterologous gene. A deletion of 9-25 bpexists at the beginning of the viral 3′NTR in CNP/LP2 and three otherfully sequenced JEV strains (Williams et al., J. Gen. Virol., 2000, 81,2471-2480; Nam et al., Am. J. Trop. Med. Hyg., 2001, 65, 388-392; Jan etal., Am. J. Troop. Med. Hyg., 1996, 55, 603-609), suggesting that thismay be a good site to insert the foreign genes. Thus, the infectious JEVcDNA developed by the present invention can act as a vector for rapidexpression of heterologous genes in a variety of cells includingmammalian cells.

VII. The present invention provides a variety of strategies forexpressing heterologous genes using the JEV-based expression vector.

It is a function of the expression vector to deliver heterologous genesof interest into cells for the expression of those genes. In the presentinvention, the full-length infectious JEV cDNA has been demonstrated toact as a heterologous gene expression vector in a variety of cell typesincluding mammalian cells.

Here, the present inventors also describe a heterologous gene expressionsystem based on the full-length infectious JEV cDNA, which serves as aBAC (Yun et al., J. Virol., 2003, 77, 6450-6465). As a transientexpression system, JEV offers several advantages: (i) high titers of thevirus are rapidly produced, (ii) the virus infects a wide range of hostcells, including insect and mammalian cell types, (iii) the geneticallystable infectious cDNA is available and readily manipulable, and (iv)the cytoplasmic replication of the RNA genome minimizes the possibilityof its integration into the host's genome and the consequent undesirablemutagenic consequences.

The present inventors demonstrated here that the JEV-based system can beused to express foreign genes in three different ways. One involvesinfectious recombinant vector RNAs/viruses encoding the foreign gene,the second involves the production of a viral replication-competent butpropagation-deficient JEV viral replicon vector RNA. The third involvesthe use of packaging systems for viral replicon particle (VRP)formation. Thus, the present inventors have shown here that the JEVsystem can be used to produce a JEV virus/infectious RNA/repliconRNA/VRP vector that will rapidly express foreign genes of interest in awide variety of mammalian cell types.

The basic method for the expression of heterologous genes using theinfectious or replicon JEV cDNA vectors of the present invention iscomposed of the following steps:

1) Preparing a recombinant JEV cDNA expression vector by insertingheterologous genes into the infectious or relicon JEV cDNA vector;

2) Producing a JEV RNA transcript from the above recombinant JEV cDNAexpression vector;

3) Preparing a transformant by transfecting host cells with the aboveJEV RNA transcript; and

4) Expressing foreign proteins by culturing the above transformant.

The present inventors produced full-length infectious recombinant JEVcDNAs expressing green fluorescent protein (GFP), an enhanced version ofGFP (EGFP), luciferase (LUC), and LacZ genes and the dominant selectivemarker puromycin N-acetyltransferase (PAC), which confers resistance tothe drug puromycin, according to the method explained hereinbefore (seeFIGS. 8 and 9). BHK-21 cells were transfected with JEV RNA transcriptstranscribed from the recombinant JEV cDNAs. GFP, EGFP, LUC, LacZ and PACexpression is shown in FIGS. 8 and 10. In addition, recombinantinfectious JEV viral particles containing those heterologous genes wereprepared from culture supernatants. The expression of those heterologousgenes was further investigated after infecting various animal cell lines(BHK-21, Vero, NIH/3T3, ST, HeLa, MDCK, CRFK, B103 and SHSY-5Y), whichhave been generally used in the field of biology and medicine, with therecombinant viruses. As a result, GFP or LUC gene inserted in virusgenome was expressed in all cells tested (see Table 4). Thus, it wasconfirmed that recombinant JEV cDNAs, JEV RNA transcripts, andrecombinant JEV viral paticles could be effectively used as a vector forexpression of foreign heterologous genes in a variety of cell types.

To independently express foreign genes using the JEV RNA replicationmachinery, the present inventors generated a panel of self-replicatingself-limiting viral replicons by deleting one, two, or all of the viralstructural genes, which meet stringent safety concerns (FIG. 11A). Theseviral replicons were capable of initiating replication and geneexpression upon RNA transfection (see FIGS. 11B and 11C).

The utility of the JEV replicon-based expression vectors was furtherelaborated by developing a panel of stable replicon packaging cell lines(PCLs) that would constitutively express all JEV viral structuralproteins (C, prM, and E) in trans (see FIG. 12). These PCLs allowed thetrans-complementation of the efficient packaging of JEV viral replicons.Thus, these PCLs were shown to be useful for efficiently producing hightiter viral VRPs upon introducing JEV viral replicons (see FIG. 12).

The present inventors also showed that infectious JEV recombinant viralRNAs encoding heterologous genes up to 3 kb can be packaged into theviral particles. By the choice of JEV viral replicon vectors such asJEV/Rep/ΔC+ΔprM+ΔE and JEV/Rep/NS1, it was estimated that a foreign geneof at least 5 kb could be packaged into the JEV VRPs. It will be ofinterest to examine the upper size limit of the foreign sequences thatcan be packaged in the JEV virion. This may be an important issue if onewishes to express lengthy genes such as cystic fibrosis transmembraneconductance regulator, whose coding sequence is approximately 4.5 kb(Flotte et al., J. Biol. Chem., 1993, 268, 3781-3790). In addition, alarge packaging capacity of JEV viral replicons would be useful if onewishes to add two or more expression units (Thiel et al., J. Virol.,2003, 77, 9790-9798; Agapov et al., Proc. Natl. Acad. Sci. USA, 1998,95, 12989-12994). In the case of the adeno-associated virus-basedvector, its packaging capacity has been elegantly expanded to bypass itsnatural size limitation (Duan et al., Nat. Med., 2000, 6, 595-598; Yanet al., Proc. Natl. Acad. Sci. USA, 2000, 97, 6716-6721), which showsthat it may be possible to expand the packaging capabilities of JEVviral replicons in a similar manner.

As with other RNA virus-derived vectors (Agapov et al., Proc. Natl.Acad. Sci. USA, 1998, 95, 12989-12994; Pushko et al., Virology, 1997,239, 389-401; Berglund et al., Nat. Biotechnol., 1998, 16, 562-565;Basak et al., J. Interferon Cytokine Res., 1998, 18, 305-313; Barclay etal., J. Gen. Virol., 1998, 79, 1725-1734; Khromykh and Westaway, J.Virol., 1997, 71, 1497-1505; Molenkamp et al., J. Virol., 2003, 77,1644-1648; Shi et al., Virology, 2002, 296, 219-233; Varnavski andKhromykh, Virology, 1999, 255, 366-375; Perri et al., J. Virol., 2000,74, 9802-9807; Curtis et al., J. Virol., 2002, 76, 1422-1434), thepresent inventors could also engineer a variety of JEV viral repliconvector RNAs that can be packaged when the structural proteins aresupplied in trans by using the alphavirus-based expression system(Agapov et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 12989-12994).Thus, the ability of packaging systems to efficiently generate biosafeJEV vectors has clearly been demonstrated. Unlike alphaviruses (Frolovaet al., J. Virol., 1997, 71, 248-258; White et al., J. Virol., 1998, 72,4320-4326) and retroviruses (Rein, Arch. Virol. Suppl., 1994, 9,513-522), little is known about the packaging signals employed byflaviviruses, including JEV. Our trans-complementation system for JEVprovides evidence that suggests the whole JEV structural region isunlikely to play a role in packaging. Thus, this system will be usefulin defining the packaging signals in JEV RNA and the regions in thestructural proteins that are involved in RNA encapsidation andmorphogenesis. This information will further enhance the utility of ourJEV-based expression systems.

In summary, the full-length JEV genomic RNA and the infectious JEV cDNAtherefrom of the present invention are not only able to identifyneurovirulence- and pathogenesis-related JEV genes but also availablefor the study of molecular mechanisms of JEV replication, transcriptionand translation. In addition, the full-length JEV genomic RNA and theinfectious JEV cDNA can be effectively used for the development oftreatment agents, vaccines, diagnostic reagents and diagnostic kits forJEV, and an expression vector for heterologous genes of interest ineukaryotic cells as well. Furthermore, the JEV-based vector systemdescribed in the present invention is a promising system by whichforeign genes can be delivered into cells in vitro and possibly in vivofor DNA immunization and transient gene therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

The application of the preferred embodiments of the present invention isbest understood with reference to the accompanying drawings, wherein:

FIG. 1 is a set of photographs showing the comparison oflarge-plaque-forming JEV isolate CNU/LP2 and original K87P39 strain.(A-B) A set of photographs showing plaque morphology using BHK-21 cells(A) or Vero cells (B). BHK-21 (A) or Vero (B) cells were mock infected(Mock-infected) or infected with the original JEV K87P39 strain(K87P39-infected), which formed a heterogeneous mixture of viral plaquesizes. The CNU/LP2 isolate purified in the present invention formed ahomogeneous population of large plaques (CNU/LP2-infected). (C) Levelsand patterns of JEV protein expression. BHK-21 cells were mock infectedor infected with K87P39, CNU/LP2 or the yellow fever virus strain YF17D.Eighteen hours later, they were fixed and stained with JEV-specificmouse hyperimmune ascites followed by fluoresceinisothiocyanate-conjugated goat anti-mouse immunoglobulin G (greenfluorescence) and confocal microscopy. Nuclei were visualized bystaining with propidium iodide (red fluorescence) in the presence ofRNase A.

FIG. 2 is a set of diagrams and a pair of electrophoresis photographsshowing strategies used to sequence genomic RNA of CNU/LP2. (A) Aschematic diagram showing the RT-PCR amplification of three overlappingcDNA amplicons representing the entire JEV genomic RNA apart from the 5′and 3′ termini. RNA is indicated in gray, and cDNA is indicated by solidparallel lines. The top panel schematically depicts the CNU/LP2 JEVgenomic RNA (10,968 base pairs in length). The bottom panels portray thethree overlapping cDNAs, JVF (nt 1 to 3865), JVM (nt 3266 to 8170), andJVR (nt 7565 to 10893). (B) A schematic diagram showing the procedure tosequence the 3′ end of CNU/LP2 genomic RNA. The 5′-phosphorylated and3′-blocked oligonucleotide T (Oligo T) was ligated to the 3′ end of JEVgenomic RNA by T4 RNA ligase, and the resulting RNA was then used forcDNA synthesis and amplification with the primers indicated by arrows.The resulting products were cloned and sequenced. (C) An electrophoresisphotograph showing the JEV-specific amplicons synthesized from theoligonucleotide T-ligated JEV genomic RNA described in (B). First-strandcDNA was synthesized with oligonucleotide TR, complementary tooligonucleotide T, and the RT reaction was carried out in the presence(lane 1) or absence (lane 2) of Superscript II reverse transcriptase.The cDNA was amplified with oligonucleotide TR and primer J35, which iscomplementary to nt 10259 to 10276. The expected size of the PCR productis 727 base pairs. The products were separated on a 1.2% agarose gel andvisualized by staining with ethidium bromide (EtBr). (D) A schematicdiagram showing the procedure to sequence the 5′ end of CNU/LP2 genomicRNA. The cap structure of viral genomic RNA was removed with tobaccoacid pyrophosphatase, and the decapped viral RNA was then self-ligatedwith T4 RNA ligase and used for cDNA synthesis and amplification. Theresulting amplified products were cloned and sequenced. (E) Anelectrophoresis photograph showing the JEV-specific ampliconssynthesized from the self-ligated JEV genomic RNA described in (D).First strand cDNA synthesis was carried out with primer J40, which iscomplementary to nt 215 to 232. The RT reaction was performed in thepresence (lane 1) or absence (lane 2) of Superscript II reversetranscriptase. The cDNA was amplified with primer J35 and primer J39,which is complementary to nt 164 to 181. The expected size of the PCRproduct is 890 base pairs. The amplified products were separated on a1.2% agarose gel and visualized by staining with EtBr. Lane M indicatesa 100-bp DNA size ladder marker (in base pairs).

FIG. 3 is a set of diagrams showing the construction of full-length JEVcDNA clones in bacterial artificial chromosome (BAC) pBeloBAC11. (A) Aschematic diagram of the full-length JEV cDNAs constructed inpBeloBAC11. Viral proteins are shown with thick solid lines at bothtermini representing the 5′ and 3′ NTRs of the viral genome. The SP6 andT7 promoter transcription start sites and the unique restrictionendonuclease recognition site ensuring runoff transcription are shown atthe 5′ and 3′ ends, respectively. (B-C) A set of schematic diagramsshowing the 5′ and 3′ termini of full-length JEV cDNA clones. Nucleotidesequences of JEV genomic RNA are shown as bold italic lowercase letters.Illustrated are the 5′ termini of four SP6-driven (B) and four T7-driven(C) full-length JEV cDNA templates (SEQ. ID. No: 68). To produce SP6 andT7 RNA polymerase runoff products, the 3′ termini of two SP6-driven (B,pBAC^(SP6)/JVFL/XhoI and pBAC^(SP6)/JVFLx/XhoI) and two T7-driven (C,pBAC^(T7)/JVFL/XhoI and pBAC^(T7)/JVFLx/XhoI) JEV cDNA templates werelinearized by Xho I digestion (SEQ. ID. No: 69), resulting in threenucleotides (CGA) of virus-unrelated sequence at the 3′ ends. Similarly,the cutting of the 3′ termini of an SP6-driven (B,pBAC^(/SP6)/JVFLx/XbaI) and a T7-driven (C, pBAC^(T7)/JVFLx/XbaI) JEVcDNA template with Xba I resulted in four nucleotides (CTAG) ofvirus-unrelated sequence at the 3′ ends (SEQ. ID. NO: 70). In contrast,the authentic 3′ end of JEV genomic RNA was present when SP6-driven (B,pBAC^(SP6)/JVFLx/XbaI^(MBN)) and T7-driven (C,pBAC^(T7)/JVFLx/XbaI^(MBN)) JEV cDNA templates were linearized by Xba Idigestion and then treated with mung bean nuclease (MBN) to remove theunrelated single-stranded sequences (SEQ. ID. No: 71). Underlined is therestriction endonuclease recognition site introduced at the 3′ end ofthe viral genome. An arrowhead indicates a cleavage site.

FIG. 4 is a set of a photograph and a graph showing the fact thatfull-length JEV cDNA template alone is not infectious but is requiredfor the generation of infectious synthetic RNAs during in vitrotranscription. (A) An electrophoresis photograph showing the cDNAtemplate and synthetic RNA transcripts. (B) A graph showing infectivityobtained by transfecting BHK-21 cells with an in vitro transcriptionreaction mixture, which contains full-length JEV cDNA template andsynthetic RNA transcripts. pBAC^(SP6)/JVFLx/XbaI (100-200 ng) linearizedwith Xba I and treated with MBN was used for SP6 polymerasetranscription in the absence (A, lane 1; B, Without Treatment) orpresence (A, lane 2; B, Dnase I During) of DNase I. After synthesis, thetranscription reaction mixture was treated for 30 min at 37° C. withDNase I (A, lane 3; B, Dnase I After) or RNase A (A, lane 4; B, Rnase AAfter). As a control, the reaction was carried out in the absence of SP6RNA polymerase (A, lane 5; B, Without SP6 Pol). (A) Following treatment,5% of the reaction mixture was separated on a 0.6% agarose gel and thecDNA template and RNA transcripts were visualized by staining with EtBr.(B) The reaction mixtures were used to transfect BHK-21 cells, andinfectious centers of plaques were estimated.

FIG. 5 is a set of photographs and graphs showing the comparison ofsynthetic JEVs with parental virus CNU/LP2. (A) Representative plaqueassays of synthetic JEVs and parent CNU/LP2. BHK-21 cells were infectedwith parent or synthetic viruses, overlaid with agarose, and stained 3days later with crystal violet. (B) Growth kinetics in BHK-21 cells ofsynthetic JEVs and parent CNU/LP2 infected at multiplicities ofinfection (MOI) of 0.01, 1, and 10. Viruses were harvested at the hourpostinfection (h.p.i) indicated, and titers were determined by plaqueassays. (C-D) Viral protein and RNA levels were analyzed byimmunoblotting (C) and Northern blotting (D), respectively. BHK-21 cellswere infected at an MOI of 1 with synthetic JEVs (lanes 1-4) or CNU/LP2(lane 5) or mock-infected (lane 6) and cultured for 18 hrs. (C) Proteinextracts were prepared from approximately 3×10⁴ cells and separated on10% SDS-polyacrylamide gels. Viral proteins were visualized byimmunoblotting with JEV-specific mouse hyperimmune ascites (top panel).In parallel, actin protein was detected as a loading and transfercontrol (bottom panel). The positions of viral protein-related cleavageintermediates and actin are indicated with arrowheads on the left.Molecular mass markers in kDa are indicated on the right. (D) Total RNAfrom approximately 1×10⁵ cells was extracted and analyzed by Northernblotting using a ³²P-labeled antisense riboprobe hybridizing to thesequence in the NS5 gene encompassing nt 9143-9351 (top panel).Etbr-stained 18S rRNA bands are shown as a loading control (bottompanel). Full-length genomic viral RNA (11 kb) and 18S rRNA are indicatedon the left.

FIG. 6 is a set of diagrams and an electrophoresis photograph showingthe presence of Xho I genetic marker in recombinant JEVs derived frompBAC^(SP6)/JVFLx/gm/XbaI. (A) Schematic diagram of the RT-PCR fragmentsof JVFLx/XbaI^(MBN) and JVFLx/gm/XbaI^(MBN) expected after Xho Idigestion. Indicated are the primers used for RT-PCR (arrows), theintroduced Xho I site (asterisk), and the sizes of the RT-PCR products(2,580 bp) and the two Xho I digestion products (1,506 bp and 1,074 bp)expected after digestion of JVFLx/gm/XbaI^(MBN) with Xho I. (B) BHK-21cells were transfected with synthetic RNAs transcribed from eitherpBAC^(SP6)/JVFLx/XbaI^(MBN) or pBAC^(SP6)/JVFLx/gm/XbaI^(MBN). Viruseswere recovered 24 hr later and serially passaged in BHK-21 cells at amultiplicity of infection of 0.1. At each passage prior to the nextround of infection, viruses were incubated with DNase I and RNase A. Atpassage 1 and 3, viral RNA was extracted from the culture supernatantcontaining the released viruses and used for RT-PCR. The PCR productswere incubated in the presence (+) or absence (−) of Xho I, separated ona 1% agarose gel, and stained with EtBr. The expected sizes of theundigested and digested PCR products are shown on the left. Lane Mindicates a 1-kb DNA ladder marker (in base pairs).

FIG. 7 is a graph showing the specific infectivity of synthetic RNAstranscribed from infectious JEV cDNA clones (pBAC^(SP6)/JVFLx/XbaI)propagated for 180 generations. Two independent clones carryingpBAC^(SP6)/JVFLx/XbaI (solid and open circles) were cultivated at 37° C.overnight in 2×YT with chloramphenicol. The primary cultures werepropagated every day for nine days by 10⁶-fold dilution and adding freshbroth for overnight growth. Each passage was estimated to be about 20generations. At the indicated passages, the DNA plasmids were purified,linearized by Xba I digestion and treated with MBN, and used astemplates for runoff transcription using SP6 RNA polymerase. Thetranscripts were then used to transfect BHK-21 cells to determinespecific infectivity.

FIG. 8 is a set of diagrams, photographs, and a graph showing theexpression of foreign genes with JEV cDNA as a vector. (A) Schematicdiagram of the cDNA templates used for runoff transcription with SP6 RNApolymerase. Indicated are the encephalomyocarditis virus (EMCV) internalribosome entry site (IRES)-driven GFP or LUC genes that were inserted atthe beginning of the 3′NTR of the viral genome, the SP6 promotertranscription start, and the runoff site generated by Xba I digestionand MBN treatment (XbaI/MBN). In pBAC^(SP6)/JVFLx/LUC^(REP-)/XbaI^(MBN),a solid vertical bar indicates an 83-nucleotide deletion (nt 5580-5663)in the middle of the NS3 gene that preterminates viral translation at nt5596 (asterisk). (B) Expression of GFP protein. BHK-21 cells weremock-transfected (Mock) or transfected with 2 μg of synthetic RNAstranscribed from the pBAC^(SP6)/JVFLx/GFP/XbaI^(MBN) template(JVFLx/GFP/XbaI^(MBN)), incubated for 30 hr, and then fixed and examinedby confocal microscopy. (C) Induction of LUC protein. BHK-21 cells(8×10⁶) were mock-transfected or transfected with 2 μg of synthetic RNAstranscribed from the pBAC^(SP6)/JVFLx/LUC/XbaI^(MBN) (●) orpBAC^(SP6)/JVFLx/LUC^(REP-)/XbaI^(MBN) (∘) templates, and seeded in a6-well plate at a density of 6×10⁵ cells per well. Cells were lysed atthe indicated time points and LUC activity was determined. The standarddeviations obtained from three independent experiments are indicated byerror bars.

FIG. 9 shows the construction and characterization of heterologousgene-encoding infectious recombinant JEVs that are based on thebicistronic full-length infectious JEV cDNA that serves as a BAC. (A)Strategy to construct the infectious recombinant JEV cDNAs. Thestructure of the parental infectious JEV cDNA (pJEV/FL) is shown (Yun etal., J. Virol., 2003, 77, 6450-6465). The viral ORFs are illustrated bythick solid lines at both termini that indicate the 5′ and 3′ NTRs ofthe viral genome. The additional expression unit driven by the EMCV IRESwas inserted at the beginning of the 3′NTR using the unique natural NsiI site. Indicated are the SP6 promoter transcription start site (SP6promoter) and the runoff site generated by Xba I digestion and MBNtreatment (XbaI/MBN). X indicates a foreign gene of interest. (B) Thestructures of the infectious recombinant JEV cDNAs constructed in thepresent invention are shown. Three commonly used reporters (EGFP, 768bp; LUC, 1653 bp; and LacZ, 3012 bp) or a dominant selective marker PAC(600 bp) were engineered to be at the beginning of the 3′NTR. In case ofthe replication-competent pJEV/FL/LUC cDNA, the replication-incompetentpJEV/FL/LUC^(REP-) cDNA was also used as a negative control byintroducing an 83-nucleotide deletion (▪) in the middle of the NS3 gene,which results in the premature termination of viral translation at nt5596 (*) as previously described (Yun et al., J. Virol., 2003, 77,6450-6465). (C-D) Comparison of the infectiousness of the recombinantJEVs with that of the parent. BHK-21 cells (8×10⁶) were mock-transfectedor transfected with 2 μg of the parent or recombinant JEV RNAs that hadbeen transcribed from the relevant JEV cDNA, as indicated. (C)Representative plaques. The transfected cells were overlaid with agaroseand stained 5 days later with crystal violet. (D) Viral proteinaccumulation. The transfected cells (4×10⁵) were lysed with 1× sampleloading buffer at the indicated time points and the protein extractswere resolved on 10% SDS-polyacrylamide gels. The viral proteins werevisualized by immunoblotting with JEV-specific mouse hyperimmune sera(Yun et al., J. Virol., 2003, 77, 6450-6465). The positions of the viralproteins (E and NS1) and the cleavage-related intermediates areindicated by arrowheads on the left. Molecular mass markers in kDa areindicated on the right. V indicates JEV CNU/LP2-infected BHK-21 cellsand N indicates naïve BHK-21 cells.

FIG. 10 shows the expression of the commonly used reporter genes and adominant selective marker using infectious JEV cDNA as the vector.BHK-21 cells (8×10⁵) were mock-transfected or transfected with 2 μg ofthe parent or recombinant JEV RNAs that had been transcribed from eachplasmid. (A-B) pJEV/FL/EGFP, (C) pJEV/FL/LacZ, (D) pJEV/FL/LUC orpJEV/FL/LUC^(REP-), (E) pJEV/FL/PAC. (A-B) Expression of EGFP. Thetransfected cells were prepared 36 hr posttransfection for confocalmicroscopy (A) and flow cytometric analysis (B). — indicates JEV/FL/EGFPRNA-transfected cells and . . . indicates mock-transfected cells. (C)Expression of LacZ. The transfected cells were processed for X-galstaining 36 hr posttransfection. (D) Induction of LUC. The transfectedcells were seeded on six-well plates at a density of 4×10⁵ cells perwell. At the indicated time points, the cell lysates were subjected toLUC assays. The experiments were done in triplicate and the mean valuesare shown by error bars. ● indicates JEV/FL/LUC RNA-transfected cells, ∘indicates JEV/FL/LUC^(REP-) RNA-transfected cells, and — indicates thelevel of background luminescence of naïve cells. (E) Expression of PAC.The transfected cells were plated on a 6-well plate and incubated incomplete media (dishes 1, 3, 5, and 7) or under a 0.5%agarose-containing overlay (dishes 2, 4, 6, and 8). After 2 daysincubation, the plates were incubated for an additional 3 days in thepresence of 10 μg/ml puromycin (dishes 5-8) or in its absence (dishes1-4). The cells were then fixed and stained with crystal violet.

FIG. 11 shows the construction and vector characteristics of JEV viralreplicons. (A) The structures of the JEV viral replicons are shown.Solid boxes (▮) indicate in-frame deletions that had been introducedinto the genome of the infectious pJEV/FL/LUC construct. Fourconstructs, namely, pJEV/Rep/ΔCC/LUC, pJEV/Rep/ΔC/LUC,pJEV/Rep/ΔprM/LUC, and pJEV/Rep/ΔE/LUC, contain a single in-framedeletion in each structural gene of JEV. pJEV/Rep/ΔCC/LUC has a deletionthat extends to the proposed cyclization sequence motif in the 5′ regionof the C gene, unlike pJEV/Rep/ΔC/LUC. Three constructs, namely,pJEV/Rep/ΔC+ΔprM/LUC, pJEV/Rep/ΔC+ΔE/LUC, and pJEV/Rep/ΔprM+ΔE/LUC,contain double in-frame deletions, while pJEV/Rep/ΔC+ΔprM+ΔE/LUC bearstriple in-frame deletions in all of the structural proteins. Alsoengineered was pJEV/Rep/NS1/LUC, which encodes the 35 N-terminal and 24C-terminal amino acids of the C protein followed immediately by theN-terminus of the NS1 protein and the rest of the viral genome. (B)Induction of LUC. Naïve BHK-21 cells (8×10⁶) were transfected with 2 μgof the parent or JEV viral replicon RNAs that had been transcribed fromeach plasmid and then seeded on 6-well plates at a density of 4×10⁵cells per well. At the indicated time points, the cell lysates weresubjected to LUC assays. The experiments were performed in triplicateand the mean values are shown. ● black, pJEV/FL/LUC; ♦ black,pJEV/FL/LUC^(REP-); ♦ blue, pJEV/Rep/ΔCC/LUC; ▪ blue, pJEV/Rep/ΔC/LUC; ▴blue, pJEV/Rep/ΔprM/LUC; ● blue, pJEV/Rep/ΔE/LUC; ▪ red,pJEV/Rep/ΔC+ΔprM/LUC; ▴ red, pJEV/Rep/ΔC+ΔE/LUC; ● red,pJEV/Rep/ΔprM+ΔE/LUC; ▪ green, pJEV/Rep/ΔC+ΔprM+ΔE/LUC; ● green,pJEV/Rep/NS1/LUC. — indicates the level of background luminescence ofnaïve cells. (C) Viral protein accumulation. The transfected cells(4×10⁵) were lysed with 1× sample loading buffer 48 hr posttransfectionand the protein extracts were resolved on 10% SDS-polyacrylamide gels.The proteins were transferred onto the nitrocellulose membrane andimmunoblotted with JEV-specific mouse hyperimmune sera.

FIG. 12 shows the construction of the packaging system for JEV viralreplicons. (A) Structures of the JEV structural protein expressioncassettes based on the Sindbis virus-based expression vector. pSinRep19is the double subgenomic noncytopathic RNA vector. A foreign gene andthe PAC gene are expressed by using separate subgenomic promoters, asindicated by arrows. The pSinRep19/JEV C-E cassette encodes the JEV C,prM, and E genes. The pSinRep19/JEV C-E-BglII cassette encodes the JEVC, prM, and E genes, followed by the N terminal 58 residues of NS1,whereas the pSinRep19/JEV C-NS1 bears a remnant of the NS1 gene. MCSindicates multiple cloning sites. (B) Western blot analysis of the JEVstructural proteins expressed from three JEV structural proteinexpression cassettes. The BHK-21 cells were mock-transfected ortransfected with each JEV structural protein expression vector RNA andlysates were obtained 48 hr later. Equivalent amounts of cell lysateswere resolved by SDS-PAGE and probed with the JEV-specific hyperimmunesera. Indicated are the positions of viral proteins E and NS1 on theright and the molecular mass markers in kDa on the left. (C) Schematicrepresentation showing how JEV VRPs can be generated by (i)co-transfection of the JEV structural protein expression vector RNAswith JEV viral replicon RNAs or (ii) transfection of the JEV structuralprotein-expressing PCLs with JEV viral replicon RNAs. (D-E) Theproduction of JEV VRPs. Two approaches were taken. One approach isinvolved the cotransfection of naïve BHK-21 cells with two vector RNAs,namely, JEV structural protein expression vector RNA and the JEV viralreplicon vector RNA indicated (D). The other approach involved JEV PCLs,which were transfected with the JEV viral replicon vector RNA indicated(E). The JEV viral replicon RNAs used were as follows: □ green,JEV/Rep/ΔC+ΔprM+ΔE/EGFP; ▪ green, JEV/Rep/NS1/EGFP; □ blue,JEV/Rep/ΔC+ΔprM+ΔE/LacZ; ▪ blue, JEV/Rep/NS1/LacZ; □ black,JEV/Rep/ΔC+ΔprM+ΔE/LUC; ▪ black, JEV/Rep/NS1/LUC. The supernatants werecollected 48 hr posttransfection and used to infect naïve BHK-21 cellsfor the titration of VRPs and the examination of the respective reportergene expression. — indicates the level of background luminescence ofnaïve cells.

EXAMPLES

Practical and presently preferred embodiments of the present inventionare illustrative as shown in the following Examples.

However, it will be appreciated that those skilled in the art, onconsideration of this disclosure, may make modifications andimprovements within the spirit and scope of the present invention.

Example 1 Isolation of JEV Viruses

<1-1> Cell Lines and Viruses

BHK-21 cell line was provided from Dr. Charles M. Rice of theRockefeller University, and maintained in alpha minimal essential medium(MEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine,vitamins, and antibiotics. All reagents used in cell culture werepurchased from Gibco/BRL Life Technologies, Inc., Gaithersburg, Md. TheKorean JEV strain K87P39 (Chung et al., Am. J. Trop. Med. Hyg., 1996,55, 91-97) was obtained from the Korean National Institute of Health.This JEV K87P39 was isolated from wild mosquitoes in Korea in 1987 andunderwent five passages in suckling mouse brains. The YF17D yellow fevervirus strain was generated from the infectious cDNA pACNR/YF17D(provided from Dr. Charles M. Rice) by SP6 polymerase runofftranscription as described bellow.

<1-2> Plaque Purification

Cells infected with the JEV K87P39 strain were overlaid with MEMcontaining 10% fetal bovine serum and 0.5% SeaKem LE agarose (FMCBioProducts, Rockland, Me.) and incubated in a 5% CO₂, 37° C. incubatorfor 3 to 4 days. After being cultured for 3 to 4 days, the infectedcells were fixed with 3.7% formaldehyde at room temperature for 4 hr.Then, agarose covering the cells was removed. Plaques were visualized bycrystal violet staining. As a result, K87P39 strain formed aheterogeneous mixture of viral plaque sizes (FIG. 1A, K87P39-infected).

Consequently, the present inventors performed the plaque purificationassay with BHK-21 cells to isolate a homogeneous population of alarge-plaque-forming variant that the present inventors named CNU/LP2.BHK-21 cells infected with the JEV K87P39 strain were overlaid with MEMcontaining 10% fetal bovine serum and 0.5% SeaKem LE agarose andincubated in a 5% CO₂, 37° C. incubator for 3 to 4 days. Individualplaques were picked with sterile Pasteur pipettes and resuspended in 1Ml of MEM. Viruses were eluted from the agarose at 4° C. for 2 hr. Theeluate was amplified only once in BHK-21 cells and stored at −80° C.

Plaque assay was performed to compare the viral plaque sizes ofsusceptible BHK-21 cells infected with JEV K87P39 and JEV CNU/LP2strains. As a result, the viral plaque sizes of susceptible BHK-21 cellsinfected with K87P39 varied (FIG. 1A, K87P39-infected). On the otherhand, the CNU/LP2 purified in the present invention formed a homogeneouspopulation of large plaques (FIG. 1A, CNU/LP2-infected). In addition,similar plaque morphologies were also observed when Vero cells wereinfected with JEV K87P39 and JEV CNU/LP2 strains (FIG. 1B).

<1-3> Immunofluorescence

In order to examine JEV expression in infected BHK-21 cells by confocalmicroscopy, cells (2×10⁵) were seeded in a four-well chamber slide,incubated for 12 hr, and then mock-infected or infected at an MOI of 1for 18 hr with either the original JEV K87P39 strain, the JEV CNU/LP2isolate, or the YF17D strain. Immunostaining for JEV viral proteins wasaccomplished by first fixing the cells by incubation inphosphate-buffered saline (PBS) containing 0.37% (v/v) formaldehyde for30 min at 25° C. The cells were then washed three times with PBS andpermeabilized for 10 min at 37° C. with PBS containing 0.2% (v/v) TritonX-100. Thereafter, the cells were washed four times with PBS, rehydratedin PBS for 15 min, and blocked for 1 hr at 37° C. with PBS containing 5%(w/v) bovine serum albumin (BSA). The cells were then incubated for 2 hrat 25° C. with 1:500-diluted mouse hyperimmune ascites fluid specificfor JEV, washed three times with PBS, incubated for 2 hr at 25° C. with1:500-diluted FITC-conjugated goat anti-mouse IgG (JacksonImmunoResearch Labs Inc.), and washed again three times with PBS.Thereafter, the cells were incubated for 30 min at 37° C. in PBScontaining 5 μg/Ml of propidium iodide and 5 μg/Ml of RNase A tolocalize the nuclei and mounted with 0.2 ml of 80% glycerol. Images wereacquired on a Zeiss Axioskop confocal microscope equipped with a 63×objective with a Bio-Rad MRC 1024 and LaserSharp software.

Confocal microscopy with anti-JEV hyperimmune ascites revealed thatCNU/LP2-infected BHK-21 cells expressed JEV viral proteins around theperinuclear membranes (FIG. 1C, CNU/LP2-infected), similar toK87P39-infected cells (FIG. 1C, K87P39-infected). This fluorescencestaining was not observed in mock-infected BHK-21 cells (FIG. 1C,Mock-infected). As a negative control, BHK-21 cells infected with yellowfever virus 17D, a flavivirus closely related to JEV, did not stain withanti-JEV hyperimmune ascites (FIG. 1C, YF17D-infected). CNU/LP2infection of a variety of animal cell lines, including the neuronalSHSY-5Y(human) and B103 (mouse) cell lines and the normeuronalVero(monkey) and MDCK (dog) cell lines, resulted in high virus titers(10⁶-10⁷ PFU/Ml) in the culture supernatants. Thus, the presentinventors decided to use CNU/LP2 as the parental strain for developing areverse genetics system for JEV.

Example 2 Complete Nucleotide Sequence Analysis of JEV CNU/LP2 GenomicRNA

Viral genomic RNA was extracted from 100 μl of virus-containing culturefluid with 300 μl of TRIzol LS reagent as recommended by themanufacturer (Gibco/BRL) and then resuspended in 20 μl of RNase-freewater. To analyze the complete nucleotide sequence of the viral genomicRNA, five overlapping cDNAs (JVF, JVM, JVR, JV3NTR, and JV35NTR)representing the entire viral RNA genome were amplified by long RT-PCR(FIG. 2). Oligonucleotides used for cDNA synthesis and amplificationwere designed according to the consensus sequence of all 16 fullysequenced JEV RNA genomes available from the GenBank database (CH2195LA,CH2195SA, FU, GP78, HVI, JaGArOl, JaOArS982, K94P05, Vellore P20778, p3,SA(A), SA(V), SA14, SA14-14-2, TC, and TL strains).

<2-1> Nucleotide Sequence Analysis of JEV CNU/LP2 Genomic RNA

For JVF amplicons (nt 1-3865), primer J7, represented by SEQ. ID. No 1and complementary to nt 3986-4003 of the JEV genome, was used for cDNAsynthesis (FIG. 2A). The primers for PCR amplification were primer J8represented by SEQ. ID. No 2 and complementary to nt 1-18, and primer J6represented by SEQ. ID. No 3 and complementary to nt 3845-3865. For JVMamplicons (nt 3266-8170), primer J4, represented by SEQ. ID. No 4 andcomplementary to nt 8150-8170 of the JEV genome, was used for cDNAsynthesis. The primers for PCR amplification were primer J20 representedby SEQ. ID. No 5 and complementary to nt 3266-3283, and primer J4. ForJVR amplicons (nt 7565-10893), primer J1, represented by SEQ. ID. No 6and complementary to nt 10947-10967 of the JEV genome, was used for cDNAsynthesis. The primers for PCR amplification were primer J12 representedby SEQ. ID. No 7 and complementary to nt 7565-7582, and primer J2represented by SEQ. ID. No 8 and complementary to nt 10870-10893. Thestandard RT reaction was conducted in a 20-μl reaction mixturecontaining 10 μl of extracted viral RNA, 5 p mol of the appropriateprimer, 100 U of Superscript II reverse transcriptase (Gibco/BRL), 40 Uof RNaseOUT (Gibco/BRL), 0.1 mM dithiothreitol (DTT), 10 mMdeoxynucleotide triphosphate (dNTP) mix, and the RT buffer supplied bythe manufacturer (Gibco/BRL) The reaction mixture was incubated at 37°C. for 1 hr and then heated at 70° C. for 15 min. A 5-μl aliquot of theRT mixture was subsequently used for PCR amplification with Pyrobest DNApolymerase (Takara Bio Inc., Shiga, Japan) and the appropriate primerpair. The PCRs were performed with 30 cycles of denaturation at 94° C.for 30 sec, annealing at 60° C. for 30 sec, and extension at 72° C. for5 min, followed by a final extension at 72° C. for 10 min. To avoid theselection bias that can occur due to cloning, the uncloned materials ofthe amplified products were directly sequenced in both directions withan automatic 3700 DNA sequencer. Sequencing analysis with twoindependently isolated preparations of viral RNA resulted in identicalsequences.

As a result, the complete nucleotide sequence of the entire viral genomeof JEV CNU/LP2 except for 3′- and 5′-terminal regions was determined andrepresented by SEQ. ID. No 9.

<2-2> Determination of 3′-Terminal Sequence of JEV CNU/LP2 Genomic RNA

In order to sequence the 3′-terminal sequences of the JEV CNU/LP2genomic RNA, a synthetic oligonucleotide T represented by SEQ. ID. No 10was ligated to the 3′ end of the viral genomic RNA to provide aprimer-binding site for cDNA synthesis and PCR amplification (Kolykhalovet al., J. Virol., 1996, 70, 3363-3371). The 3′ end of oligonucleotide Twas first modified by incorporating ddATP with terminaldeoxynucleotidyltransferase (Takara), which blocks the intramolecularand intermolecular ligation of oligonucleotide T. The 5′ end ofoligonucleotide T was also phosphorylated with T4 polynucleotide kinase(Takara). Thereafter, the modified oligonucleotide T was ligated to the3′ end of the viral genomic RNA by T4 RNA ligase (New England Biolabs,Inc., Beverly, Mass.). The 20 μl of ligation reaction mixture contained10 U of T4 RNA ligase, 40 U of RNaseOUT, 10 p mol of oligonucleotide T,viral genomic RNA, and the buffer supplied by the manufacturer (NEB).After incubation at 16° C. for 12 hr, the ligated viral RNA was phenolextracted, precipitated with ethanol, and resuspended with 20 μl ofRNase-free water. Subsequently, 10 μl of the oligonucleotide-ligatedviral RNA was used for cDNA synthesis with oligonucleotide TRrepresented by SEQ. ID. No 11, which is complementary to oligonucleotideT, as previously described. First-strand cDNA was amplified with primerJ35 represented by SEQ. ID. No 12 and complementary to nt 10259 to10276, and primer TR. For PCR, 5 μl aliquot of the RT reaction mixturewas amplified with Pyrobest DNA polymerase and 30 cycles of 30 sec at94° C., 30 sec at 60° C., and 1 min at 72° C., followed by a finalextension of 10 min at 72° C. The PCR mixtures were as described above.The cDNA amplicons designated as JV3NTR were cloned into the pRS2 vector(provided by Dr. Charles M. Rice) with Hind III and EcoR I sitesincorporated in the positive-sense and negative-sense primers,respectively (FIG. 2B).

As a result of agarose gel electrophoresis, it was revealed that theamplified products migrated as two bands, a larger band of approximately700 bp and a smaller band of about 450 bp (FIG. 2C). Both bands werepurified and cloned, and 20 and 10 randomly picked clones containing thelarger and the smaller bands, respectively, were sequenced. As has beendocumented for most of the fully sequenced JEV isolates, the presentinventors found that all the clones with the larger insert (about 700bp) terminated the viral genome with -GATCT¹⁰⁹⁶⁸. In contrast, all theclones with the smaller insert (about 450 bp) showed the viral genometruncated at nt 10684, resulting in a band 284 bp shorter. Duringassembly of the full-length JEV cDNA, the present inventors used thenucleotide sequences of the larger insert because the smaller insert didnot contain 284 nucleotides at the 3′ end of the viral genome.

<2-3> Determination of 5′-Terminal Sequence of JEV CNU/LP2 Genomic RNA

The 5′-terminal sequence of JEV CNU/LP2 genomic RNA was determined byself-ligation of viral RNA (Campbell and Pletnev, Virology, 2000, 269,225-237). The cap structure of viral genomic RNA was first cleaved offwith tobacco acid pyrophosphatase (TAP). The cleavage reaction mixture(20 μl) contained 10 U of TAP (Epicentre Technology Co., Madison, Wis.),10 μl of viral RNA, and the buffer supplied by the manufacturer(Epicentre Technology Co.). After incubation at 37° C. for 1 hr, theTAP-treated viral RNA was subjected to phenol extraction and ethanolprecipitation, and resuspended with 20 μl of RNase-free water. Half (10μl) of the decapped viral RNA was self-ligated in a 20-μl reactionmixture with T4 RNA ligase as described above. A quarter (5 μl) of theself-ligated viral RNA was used for cDNA synthesis with primer J40,represented by SEQ. ID. No 13 and complementary to nt 215 to 232.First-strand cDNA was PCR amplified with primer J39 represented by SEQ.ID. No 14 and complementary to nt 164 to 181, and primer J35 (FIG. 2D).Agarose gel electrophoresis revealed the amplified products as a singleband of about 850 bp (FIG. 2E). The amplified cDNA amplicons (JV35NTR)were digested with Apo I and Spe I, and ligated into the pRS2 vectorwhich had been digested with Apo I and Xba I, leading to constructpRS2/JV3′5′.

To sequence the 5′-terminal sequences of the JEV CNU/LP2 genomic RNA, 12randomly picked clones were sequenced. In all 12 clones, the presentinventors found that the -GATCT¹⁰⁹⁶⁸ of the viral 3′-terminal sequencewas followed by the 5′-terminal sequence ¹AGAAGT- (FIGS. 2B and 2C).Identical results were also obtained by direct cycle sequencing ofuncloned material. Thus, the present inventors have determined thecomplete nucleotide sequence of the CNU/LP2 isolate and confirmed thatthe sequence is represented by SEQ. ID. No 15.

Example 3 Construction of Full-length Infectious cDNAs for JEV

During our initial attempts to clone the cDNA of the CNU/LP2 RNA genome,it became apparent that a particular region of the viral genome was notcompatible with cloning in high-copy-number plasmids in E. coli becausethe cloned DNA underwent genetic rearrangements. These difficulties havealso been reported for other flaviviruses (Campbell and Pletnev,Virology, 2000, 269, 225-237; Polo et al., J. Virol., 1997, 71,5366-5374; Gritsun and Gould, Virology, 1995, 214, 611-618; Sumiyoshi etal., J. Infect. Dis., 1995, 171, 1144-1151; Sumiyoshi et al., J. Virol.,1992, 66, 5425-5431; Rice et al., New Biol., 1989, 1, 285-296). Attemptsto clone this region into a low-copy-number bacterial plasmid were alsounsuccessful due to genetic instability together with a low DNA yield.Thus, the present inventors used the bacterial artificial chromosome(BAC) plasmid pBeloBAC11 as a vector to house full-length infectiouscDNAs for JEV.

<3-1> Subcloning of Three Long Overlapping JEV cDNA Amplicons

The present inventors used recombinant DNA techniques according tostandard procedures (Sambrook et al., Molecular cloning, 1989, ColdSpring Harbor Laboratory). First, three overlapping cDNA amplicons (JVF,JVM and JVR) originally used for complete nucleotide sequence analysiswere subcloned into pBAC/SV represented by SEQ. ID. No 42, a derivativeof the pBeloBAC11 plasmid. The pBAC/SV plasmid contains the 491-bp NotI-Aat II (T4 DNA polymerase-treated) fragment of pACNR/NADL (Mendez etal., J. Virol., 1998, 72, 4737-4745), the 9,215-bp Sac I (T4 DNApolymeras-treated)-Ssp I (T4 DNA polymerase-treated) fragment ofpSINrep19 (Frolov et al., Proc. Natl. Acad. Sci., USA., 1996, 93,11371-11377), and the 6,875-bp Sfi I (T4 DNA polymerase-treated)-Not Ifragment of pBeloBAC11. Thus, the 3,863-bp Rsr II-Avr II fragment of theJVF amplicons, the 4,717-bp BspE I-Mlu I fragment of the JVM amplicons,and the 3,326-bp Rsr II-Bgl II fragment of the JVR amplicons wereinserted into the pBAC/SV plasmid, which had been digested with the sameenzymes. This led to the pBAC/JVF, pBAC/JVM, and pBAC/JVR subcloneconstructs, respectively. These BAC plasmids were grown in E. coli DH10Bcells and sequenced. The nucleotide sequences of the cloned cDNAs wereidentical to that of CNU/LP2 with the exception of a point mutation,T⁸⁹⁰⁶→C (silent), within the NS5 gene in pBAC/JVR. The T⁸⁹⁰⁶→Csubstitution was translationally silent and must have arisen during thecloning because sequencing of eight randomly picked individual clonesrevealed a T residue at nt 8906. Although the T⁸⁹⁰⁶→C substitution doesnot alter the corresponding amino acid, it is possible that this changecould affect viral replication (van Dinten et al., Proc. Natl. Acad.Sci. USA, 1997, 94, 991-996), and thus the present inventors correctedthis substitution back to a T residue. The T⁸⁹⁰⁶→C substitution wascorrected by recloning a 315-bp Apa I-Hind III fragment corresponding tont 8827 to 9142, leading to the construct pBAC/JVRR. During theirmanipulation and propagation in the E. coli strain DH10B, all threesubcloned JEV cDNAs remained genetically stable.

<3-2> Insertion of SP6 Promoter into the 5′ End of the Full-Length JEVcDNA

In order to facilitate the precise adjoining of the bacteriophage SP6promoter transcription start to the 5′ end of the full-length JEV cDNA,the present inventors modified the pBAC/JVF. First, two fragments wereisolated by PCR of pBAC/SV with primer J41 represented by SEQ. ID. No 16and primer J43 represented by SEQ. ID. No 17, which incorporates thenegative-sense sequence of the SP6 promoter and PCR of pBAC/JVF withprimer J42 represented by SEQ. ID. No 18 and primer J40 represented bySEQ. ID. No 19. These two fragments were fused by a second round of PCRwith primers J41 and J40. The resulting amplicons were digested with PacI and Pme I, and ligated with pBAC/JVF which had been digested with thesame two enzymes. This produced pBAC^(SP6)/JVF.

<3-3> Construction of Full-Length JEV cDNAs Containing SP6 Promoter

In order to generate an authentic or nearly authentic 3′ terminus duringrunoff transcription of plasmid linearized at the 3′ end of the viralgenome, the present inventors modified pBAC/JVRR so that the nucleotidesequence of the authentic 3′ terminus was followed by a uniquerestriction endonuclease recognition site, either Xho I or Xba I. Tocreate the pBAC/JVRR/XhoI subclone containing a unique Xho I site at theend of the viral genome, fragment I was synthesized by PCR amplificationof pRS2/JV3′5′ with primer J90 represented by SEQ. ID. No 20 and primerJ45 represented by SEQ. ID. No 21, which incorporates an Xho I site. The298-bp Sfi I-Spe I portion of fragment I amplicons was ligated withpBAC/JVRR which had been digested with Sfi I and Nhe I. To createpBAC/JVRRx/XbaI, which has an Xba I site at the end of the viral genome,the existing Xba I site at nt 9,131 to 9,136 within the NS5 gene wasfirst inactivated by introducing a silent point mutation (A⁹¹³⁴→T) byPCR. In this construct, the “x” denotes the presence of the silent pointmutation (A⁹¹³⁴→T) that destroyed the original Xba I site. Particularly,PBAC/JVRR was amplified with primer J31 represented by SEQ. ID. No 22and primer J47 represented by SEQ. ID. No 23, which incorporated theA⁹¹³⁴ →T substitution. The 315-bp Apa I-Hind III portion of the cDNAamplicons, corresponding to nt 8,828 to 9,143, was cloned intopBAC/JVRR, leading to the construct pBAC/JVRRx. Subsequently,pBAC/JVRRx/XbaI was constructed in the same manner as described forpBAC/JVRR/XhoI. Thus, fragment II was obtained by PCR amplification ofpRS2/JV3′5′ with primer J90 and primer J46 represented by SEQ. ID. No24, which incorporated an Xba I site. The 298-bp Sfi I-Spe I portion ofthe fragment II amplicons was then ligated into pBAC/JVRRx which hadbeen digested with Sfi I and Nhe I. To create pBAC/JVRRx/XhoI containinga unique Xho I site and the A⁹¹³⁴→T substitution, the 298-bp Sfi I-Spe Iportion of fragment I amplicons was ligated into pBAC/JVRRx which hadbeen digested with Sfi I and Nhe I.

Thus, the present inventors constructed five plasmids, pBAC^(SP6)/JVF,pBAC/JVM, pBAC/JVRR/XhoI, pBAC/JVRRx/XbaI, and pBAC/JVRRx/XhoI. Theseplasmids contained contiguous regions of the JEV genome and could now beused to assemble three different full-length JEV cDNAs (FIG. 3). First,the pBAC^(SP6)/JVFM subclone was constructed by ligating together the4,717-bp BspE I-Mlu I fragment of pBAC/JVM, the 8,970-bp BspE I-Xba Ifragment of pBAC^(SP6)/JVF, and the 3,670-bp Xba I-Mlu I fragment ofpBAC/SV. Subsequently, two fragments of pBAC^(SP6)/JVFM (the 8,142-bpPac I-Sap I fragment and the 4,801-bp Pac I-BsrG I fragment) wereligated with either i) the 5,620-bp Sap I-BsrG I fragment ofpBAC/JVRR/XhoI to generate pBAC^(SP6)/JVFL/XhoI, ii) the 5,622-bp SapI-BsrG I fragment of pBAC/JVRRx/XbaI to generate pBAC^(SP6)/JVFLx/XbaI,or iii) the 5,620-bp Sap I-BsrG I fragment of pBAC/JVRRx/XhoI togenerate pBAC^(SP6)/JVFLx/XhoI. Finally, three assembled full-length JEVcDNAs were designated pBAC^(SP6)/JVFL/XhoI, pBAC^(SP6)/JVFLx/XhoI, andpBAC^(SP6)/JVFLx/XbaI and represented by SEQ. ID. No 43, No 44, and No45, respectively (FIG. 3B). These cDNA clones all had the SP6 promotertranscription start at the beginning of the viral genome so thatsynthetic RNA transcripts with an authentic 5′ end would be generatedthrough in vitro transcription using SP6 RNA polymerase (FIG. 3B, graybox). To ensure that the 3′ end of the viral genome after runofftranscription would be close to authentic, the present inventors placeda unique restriction endonuclease recognition site, either Xho I or XbaI, at the end of the viral genome (FIG. 3B, underlined). Thus,pBAC^(SP6)/JVFL/XhoI bears an Xho I site at the end of the viral genome.For the construct with an Xba I site immediately at the end of viralgenome, as the viral genome already contains an Xba I site in the NS5gene, this site had to be destroyed by introducing a silent pointmutation (A⁹¹³⁴→T). This construct was designated pBAC^(SP6)/JVFLx/XbaI,where the “x” denotes the presence of the silent point mutation thatdestroyed the original Xba I site. The third clone,pBAC^(SP6)/JVFLx/XhoI, contains both the Xho I site at the end of viralgenome and the A⁹¹³⁴→T substitution.

The present inventors deposited the pBAC^(SP6)/JVFLx/XbaI at Gene Bankof Korea Research Institute of Bioscience and Biotechnology (KRIBB) onOct. 2, 2002 (Accession No: KCTC 10347 BP).

<3-4> Construction of Full-Length JEV cDNAs Containing T7 promoter

In addition to the SP6-driven JEV cDNAs, the present inventors alsoconstructed a set of three T7-driven full-length JEV cDNAs in a similarmanner of the Example <3-3>. First, a fragment from pBAC/NADLcIn-/PAC(provided by Dr. Charles M. Rice) was synthesized by PCR with the primerJ81 represented by SEQ. ID. No 25 and the primer J80 represented by SEQ.ID. No 26. A fragment from pBAC^(SP6)/JVFLx/XbaI was also synthesizedwith the primer J42 represented by SEQ. ID. No 27 and the primer J82represented by SEQ. ID. No 28. These two fragments were fused by thesecond round of PCR with the primers J81 and J82. The 793-bp EcoR I-SpeI fragment of the resulting amplicons was inserted into the pRS2 vectordigested with EcoR I and Xba I, leading to the construct pRS2^(T7)/5′JV.The 675-bp Pvu I-Pme I fragment of pRS2^(T7)/5′JV was ligated witheither i) the 18,364-bp Pac I-Pme I fragment of pBAC^(SP6)/JVFL/XhoI tocreate pBAC^(T7)/JVFL/XhoI, ii) the 18,364-bp Pac I-Pme I fragment ofpBAC^(SP6)/JVFLx/XhoI to create pBAC^(T7)/JVFLx/XhoI, or iii) 18,366-bpPac I-Pme I of pBAC^(SP6)/JVFLx/XbaI to create pBAC^(T7)/JVFLx/XbaI.Finally, three assembled full-length JEV cDNAs were designatedpBAC^(T7)/JVFL/XhoI, pBAC^(T7)/JVFLx/XhoI, and pBAC^(T7)/JVFLx/XbaI andrepresented by SEQ. ID. No 46, No 47, and No 48, respectively (FIG. 3C).At every cloning step during the assembly process, the structuralintegrity of the cloned cDNAs was assessed by extensive restriction andnucleotide sequence analyses. Structural instability of the insertsleading to deletions or rearrangements was not observed.

The present inventors deposited the pBAC^(T7)/JVFLx/XbaI at Gene Bank ofKorea Research Institute of Bioscience and Biotechnology (KRIBB) on Oct.2, 2002 (Accession No: KCTC 10346BP).

Example 4 Transcriptions and Transfections

The present inventors synthesized RNA transcripts by in vitrotranscription. Particularly, 100 to 200 ng of the template DNAlinearized with Xho I or Xba I digestion and in some cases modified withMBN was added to a 25-μl reaction mixture consisting of the buffersupplied by the manufacturer (Gibco/BRL) plus 0.6 mM cap analog[m⁷G(5′)ppp(5′)A or m⁷G(5′)ppp(5′)G, NEB Inc.], 0.5 μM [³H]UTP (1.0mCi/Ml, 50 Ci/m mol, New England Nuclear Corp., Boston, Mass.), 10 mMDTT, 1 mM each UTP, GTP, CTP and ATP, 40 U of RNaseOUT, and 15 U of SP6RNA polymerase (Gibco/BRL). The reaction mixtures were incubated at 37°C. for 1 hr. RNAs were quantified on the basis of [³H]UTP incorporationas measured by RNA adsorption to DE-81 (Whatman, Maidstone, UK) filterpaper (Sambrook et al., Molecular cloning, 1989, Cold Spring HarborLaboratory). A 1- to 1.5-μl aliquot of reaction mixture was examined byagarose gel electrophoresis, and aliquots were stored at −80° C. untiluse.

For RNA transfection, cells were electroporated with synthetic RNAs witha model ECM 830 electroporator (BTX Inc., San Diego, Calif.), asrecommended by the manufacturer. Briefly, subconfluent cells weretrypsinized, washed three times with ice-cold RNase-free PBS, andresuspended at a density of 2×10⁷ cells/Ml in PBS. A 400-μl aliquot ofthe suspension was mixed with 2 μg of synthetic RNA, and the cells wereimmediately electroporated under the conditions determined previously tobe optimal (980 V, 99-μs pulse length, and five pulses). Theelectroporated mixture was then transferred to 10 Ml of fresh medium.

An infectious center assay was used to quantify the specific infectivityof the synthetic RNA. Particularly, for runoff transcription, JEV cDNAtemplates were linearized by digestion with Xho I or Xba I. SP6polymerase runoff transcription of the two Xho I-linearized plasmids(pBAC^(SP6)/JVFL/XhoI and pBAC^(SP6)/JVFLx/XhoI) in the presence of them⁷G(5′)ppp(5′)A cap structure analog yielded capped synthetic RNAscontaining three nucleotides (CGA) of virus-unrelated sequence at their3′ ends (FIG. 3B). This is the result of copying the 5′ overhang left bythe Xho I digestion (FIG. 3B). Similarly, SP6 polymerase runofftranscription of the Xba I-linearized pBAC^(SP6)/JVFLx/XbaI plasmid inthe presence of the m⁷G(5′)ppp(5′)A cap structure analog produced cappedsynthetic RNAs with four nucleotides (CTAG) of virus-unrelated sequenceat their 3′ ends (FIG. 3B). The electroporated cells were seriallydiluted 10-fold and plated on monolayers of untransfected cells (5×10⁵)in a six-well plate. Cells were allowed to attach to the plate for 6 hr,after which they were overlaid with 0.5% SeaKem LE agarose-containingMEM as described above. The plates were incubated for 3 to 4 days at 37°C. with 5% CO₂, and infectious plaque centers were visualized by crystalviolet staining.

When susceptible BHK-21 cells were transfected with the synthetic RNAsfrom these constructs, all were highly infectious (Table 3). That is,the synthetic RNAs obtained from pBAC^(SP6)/JVFL/XhoI,pBAC^(SP6)/JVFLx/XhoI, and pBAC^(SP6)/JVFLx/XbaI transfected underoptimal electroporation conditions had specific infectivities of3.5×10⁵, 4.3×10⁵, and 3.4×10⁵ PFU/μg, respectively (Table 3,infectivity). Similar results were also obtained with synthetic RNAstranscribed from the T7-driven cDNA constructs by T7 polymerase runofftranscription (Table 3, infectivity).

TABLE 3 Specific infectivity of in vitro RNA transcripts generated fromfull-length JEV cDNAs and virus titer Infectivity^(b) Templates used for(PFU/μg of Virus titer^(c)(PFU/Ml) transcription^(a) RNA) 24 hr 48 hrpBAC^(SP6)/JVFL/XhoI 3.5 × 10⁵ 4.4 × 10⁵ 3.6 × 10⁶ pBAC^(T7)/JVFL/XhoI2.9 × 10⁵ 2.0 × 10⁵ 2.3 × 10⁶ pBAC^(SP6)/JVFLx/XhoI 4.3 × 10⁵ 2.1 × 10⁵5.2 × 10⁶ pBAC^(T7)/JVFLx/XhoI 3.8 × 10⁵ 3.3 × 10⁵ 4.1 × 10⁶pBAC^(SP6)/JVFLx/XbaI 3.4 × 10⁵ 3.5 × 10⁵ 3.2 × 10⁶ pBAC^(T7)/JVFLx/XbaI3.0 × 10⁵ 2.4 × 10⁵ 2.7 × 10⁶ pBAC^(SP6)/JVFLx/XbaI^(MBN) 3.1 × 10⁶ 6.2× 10⁶ 1.4 × 10⁶ pBAC^(T7)/JVFLx/XbaI^(MBN) 2.7 × 10⁶ 5.6 × 10⁶ 2.4 × 10⁶^(a)All full-length JEV cDNAs were linearized with an appropriaterestriction endonuclease for runoff transcription as indicated in thenames of the cDNAs. For pBAC^(SP6)/JVFLx/XbaI^(MBN) andpBAC^(T7)/JVFLx/XbaI^(MBN), these cDNA templates were prepared bylinearization with XbaI digestion, which was followed by treatment withMBN. ^(b)After in vitro transcription with SP6 or T7 RNA polymerase, asindicated, samples were used to electroporate BHK-21 cells, andinfectious plaque centers were determined. ^(c)Virus titers at 24 and 48hr postelectroporation.<4-1> Construction of JEV RNA Transcripts Lacking the Virus-unrelatedSequences at their 3′ Ends

It has been reported that for some flaviviruses, the presence ofunrelated sequences at the 3′ end of synthetic RNAs transcribed frominfectious cDNA diminishes or abrogates their specific infectivity(Yamshchikov et al., Virology, 2001, 281, 294-304). Based on thisreport, the present inventors generated synthetic RNAs lacking thevirus-unrelated sequences at their 3′ends and compared their specificinfectivities. Particularly, the present inventors generated syntheticJEV RNAs lacking the virus-unrelated sequences by treating the XbaI-linearized pBAC^(SP6)/JVFLx/XbaI plasmid with MBN prior to thetranscription reaction, which removed the four excess nucleotides ofCTAG. RNA transcripts from Xba I-linearized and MBN-treatedpBAC^(SP6)/JVFLx/XbaI and pBAC^(T7)/JVFLx/XbaI(pBAC^(SP6)/JVFLx/XbaI^(MBN), FIG. 3B and pBAC^(T7)/JVFLx/XbaI^(MBN),FIG. 3C) both had increased specific infectivities compared to theuntreated transcripts. Precisely, the specific infectivity of RNAstranscribed from pBAC^(SP6)/JVFLx/XbaI^(MBN) was estimated to be 3.1×10⁶PFU/μg, approximately 10-fold higher than the specific infectivity(3.4×10⁵ PFU/μg) of the unmodified template (Table 3, infectivity). TheRNAs derived from pBAC^(T7)/JVFLx/XbaI also had increased specificinfectivity after MBN modification (2.7×10⁶ PFU/μg) (Table 3,infectivity). Therefore, the present inventors demonstrated that theauthentic 3′ end of the JEV genome should be present to ensure highlyinfectious synthetic JEV RNA transcripts are generated.

In addition, the altered specific infectivity of the RNA transcripts dueto the presence of three or four virus-unrelated nucleotides at the 3′end also influences the virus titers harvested from culture supernatantsof the transfected BHK-21 cells. Virus titers released from BHK-21 cellstransfected with RNA transcripts from MBN-untreatedpBAC^(SP6)/JVFL/XhoI, pBAC^(SP6)/JVFLx/XhoI, and pBAC^(SP6)/JVFLx/XbaIranged from 2.1×10⁵ to 4.4×10⁵ PFU/Ml at 24 hr posttransfection (Table3, virus titer 24 hr), at which time half of the transfected cells werestill attached to culture dishes showing virus-induced strong cytopathiceffect. These titers increased about 10-fold to the range of 3.2×10⁶ to5.2×10⁶ PFU/Ml at 48 hr posttransfection (Table 3, virus titer 48 hr),at which point most of the cells had died and detached from the bottomof the culture dishes. In contrast, the virus titer released from BHK-21cells transfected with RNA transcripts from MBN-treatedpBAC^(SP6)/JVFLx/XbaI^(MBN) had already reached 6.2×10⁶ PFU/Ml at 24 hrposttransfection, at which time the majority of the transfected cellshad died (Table 3, virus titer 24 hr). This titer decreased slightly to1.4×10⁶ PFU/Me at 48 hr posttransfection (Table 3, virus titer 48 hr).Similar patterns of virus production were seen with the T7polymerase-driven RNA transcripts (Table 3).

Example 5 Confirmation of Specific Infectivity of Synthetic RNATranscripts

The present inventors confirmed that specific infectivity requires thetranscription of RNA from the full-length JEV cDNA template by using thefull-length cDNA clone pBAC^(SP6)/JVFLx/XbaI^(MBN) (FIG. 4). The cDNAtemplate alone was not infectious (FIG. 4A, lane 5 and B, without SP6Pol), but the intact cDNA template was needed during the transcriptionreaction because DNase I treatment abolished infectivity (FIG. 4A, lane2 and B, DNase I During). Addition of DNase I after the transcriptionreaction had no effect (FIG. 4A, lane 3 and B, DNase I after) relativeto the intact reaction mixture (FIG. 4A, lane 1 and B, withouttreatment), but RNase A treatment abolished the infectivity of thetranscribed synthetic RNAs (FIG. 4A, lane 4 and B, RNase A after).

Example 6 Comparison of Synthetic JEVs Recovered from Full-lengthInfectious cDNAs with the CNU/LP2 Parental Virus

The present inventors compared the synthetic JEVs recovered fromfull-length infectious cDNAs (pBAC^(SP6)/JVFL/XhoI,pBAC^(SP6)/JVFLx/XhoI, pBAC^(SP6)/JVFLx/XbaI, andpBAC^(SP6)/JVFLx/XbaI^(MBN)) with the parental virus CNU/LP2 originallyused for cDNA construction (plaque morphology, growth kinetics, proteinexpression, RNA production, etc).

<6-1> Comparison of Plaque Morphology by Plaque Assay

BHK-21 cells were infected with the synthetic JEVs recovered fromfull-length infectious cDNAs (pBAC^(SP6)/JVFL/XhoI,pBAC^(SP6)/JVFLx/XhoI, pBAC^(SP6)/JVFLx/XbaI, andpBAC^(SP6)/JVFLx/XbaI^(MBN)) and the parental virus CNU/LP2. The cellswere overlaid with MEM containing 10% fetal bovine serum and 0.5% SeaKemLE agarose (FMC BioProducts, Rockland, Me.) and incubated in a 5% CO₂,37° C. incubator for 3 to 4 days. After being cultured for 3 to 4 days,the infected cells were fixed with 3.7% formaldehyde at room temperaturefor 4 hr. Then, agarose covering the cells was removed. Plaques werevisualized by crystal violet staining. As shown in FIG. 5A, BHK-21 cellsinfected with synthetic JEVs recovered from pBAC^(SP6)/JVFL/XhoI (dish1), pBAC^(SP6)/JVFLx/XhoI (dish 2), pBAC^(SP6)/JVFLx/XbaI (dish 3), andpBAC^(SP6)/JVFLx/XbaI^(MBN) (dish 4) formed homogeneous large plaques,similar to the cells infected with CNU/LP2 (dish 5).

<6-2> Comparison of Growth Kinetics

The present inventors infected BHK-21 cells with the synthetic JEVsrecovered from full-length infectious cDNAs (pBAC^(SP6)/JVFL/XhoI,pBAC^(SP6)/JVFLx/XhoI, pBAC^(SP6)/JVFLx/XbaI, andpBAC^(SP6)/JVFLx/XbaI^(MBN)) and the parental virus CNU/LP2. BHK-21cells were infected with low (0.01 PFU/cell), medium (1.0 PFU/cell), andhigh (10 PFU/cell) MOI, after which the cell culture fluids wereharvested periodically and used to determine the kinetics of infectiousvirus release over time. Particularly, viruses were harvested at theindicated time points, and titers were determined by plaque assay. Asshown in FIG. 5B, the MOI-dependent virus titers accumulating over timewere similar for the four recovered viruses (pBAC^(SP6)/JVFL/XhoI,pBAC^(SP6)/JVFLx/XhoI, pBAC^(SP6)/JVFLx/XbaI, andpBAC^(SP6)/JVFLx/XbaI^(MBN)) and the parental virus CNU/LP2.

<6-3> Comparison of Viral Protein Level by Western Blot Analysis

The present inventors compared viral protein expressed in BHK-21 cellsinfected with the synthetic JEVs recovered from full-length infectiouscDNAs (pBAC^(SP6)/JVFL/XhoI, pBAC^(SP6)/JVFLx/XhoI,pBAC^(SP6)/JVFLx/XbaI, and pBAC^(SP6)/JVFLx/XbaI^(MBN)) with that inBHK-21 cells infected with the parental virus CNU/LP2. Particularly,BHK-21 cells (3×10⁵) were lysed with 200 μl of sample loading buffer [80mM Tri-HCl (pH 6.8), 2.0% SDS, 10% glycerol, 0.1 M DTT, 0.2% bromophenolblue], and one-tenth of the lysate was boiled for 5 min and fractionatedon an SDS-polyacrylamide gel. Proteins were transferredelectrophoretically onto a methanol-activated polyvinylidene difluoridemembrane with a Trans-Blot SD electrophoretic transfer cell machine(Bio-Rad Laboratories Inc., Hercules, Calif.), and the membrane wasblocked at room temperature for 1 hr with 5% nonfat dried milk inwashing solution (0.2% Tween 20 in PBS). After three washes with washingsolution, membranes were incubated at room temperature for 2 hr witheither a monoclonal anti-actin antibody (A4700, Sigma, St. Louis, Mo.)that recognizes the epitope conserved in the C terminus of all actinisoforms or mouse hyperimmune ascites fluid specific for JEV (ATCCVR-1259AF, American Type Culture Collection). The membranes were thenwashed three times with washing solution and incubated at roomtemperature for 2 hr with alkaline phosphatase (AP)-conjugated goatanti-mouse immunoglobulin G (Jackson ImmunoResearch Labs Inc., WestGrove, Pa.). The membranes were washed three times with washing solutionand once with PBS. Actin and JEV protein bands were visualized byincubation with the substrates 5-bromo-4-chloro-3-indolylphosphate andnitroblue tetrazolium. As a result, it was demonstrated that thesynthetic JEVs and the parental virus produced similar amounts andidentical patterns of virus-specific proteins (FIG. 5C, top panel).Actin protein was measured as an internal sample loading control andrevealed equivalent levels of actin protein in mock-infected andinfected cells (FIG. 5C, bottom panel).

<6-4> Comparison of Viral RNA Level by Northern Blot Analysis

The present inventors compared viral RNA expressed in BHK-21 cellsinfected with the synthetic JEVs recovered from full-length infectiouscDNAs (pBAC^(SP6)/JVFL/XhoI, pBAC^(SP6)/JVFLx/XhoI,pBAC^(SP6)/JVFLx/XbaI, and pBAC^(SP6)/JVFLx/XbaI^(MBN)) with that inBHK-21 cells infected with the parental virus CNU/LP2. Particularly,total RNA was extracted from infected BHK-21 cells (3×10⁵) with 1 Ml ofTRIzol reagent (Gibco/BRL). One-third of the RNA was analyzed forJEV-specific RNA by Northern blot analysis (Sambrook et al., Molecularcloning, 1989, Cold Spring Harbor Laboratory). The RNA waselectrophoresed in denaturing 2.2 M formaldehyde-1% agarose gels andtransferred onto nylon membranes (Amersham Biosciences Inc., Piscataway,N.J.). The RNA on the membranes was cross-linked by irradiation with a254-nm light source (Stratalinker UV cross-linker, Stratagene, La Jolla,Calif.), and the JEV-specific RNAs were detected by hybridization with a[³²P]CTP-labeled antisense riboprobe that binds to nt 9,143 to 9,351 ofthe JEV genome. This probe had been synthesized by in vitrotranscription from the BamH I-linearized cDNA clone pGEM3Zf(+)/JV9143,which was constructed by ligating the 209-bp Hind III-Sac I fragment ofpBAC^(SP6)/JVFLx/XbaI with pGEM3Zf(+) digested with the same enzymes.This clone was transcribed with the T7-MEGAscript kit (Ambion, Austin,Tex.) as recommended by the manufacturer with a 20-μl reaction mixturecontaining 3.12 μM [α-³²P]CTP(800 Ci/m mol, Amersham). After beingtreated with DNase I, the reaction mixture was spun in a Quick Spin G-50Sephadex column (Boehringer Mannheim) to remove unincorporatedribonucleoside triphosphates. The membrane was prehybridized at 55° C.for 6 hr in hybridization solution [5×SSPE(0.9 M NaCl, 50 mM NaH₂PO₄,and 5 mM EDTA pH 7.7), 5× Denhardt's reagent, 0.5% SDS, 100 μg/Ml ofdenatured salmon sperm DNA, 50% formamide] and then incubated at 55° C.overnight in the hybridization solution containing 10⁷ cpm of thelabeled riboprobe. The membrane was washed three times at 55° C. for 10min with 1×SSPE-0.5% SDS and once for 10 min with 0.1×SSPE-0.5% SDS.Viral RNA bands were visualized by autoradiography and quantified with aMolecular Imager (Bio-Rad Lab). As a result, viral RNA levels were allsimilar (FIG. 5D). Quantification of these blots by image analysisrevealed that the ratios of viral genomic RNA (FIG. 5D, top panel) to18S rRNA (FIG. 5D, bottom panel) did not differ significantly,demonstrating that all viral genomic RNAs were produced at similarlevels.

Thus, all the synthetic viruses recovered from full-length infectiouscDNAs (pBAC^(SP6)/JVFL/XhoI, pBAC^(SP6)/JVFLx/XhoI,pBAC^(SP6)/JVFLx/XbaI, and pBAC^(SP6)/JVFLx/XbaI^(MBN)) wereindistinguishable from the parental virus CNU/LP2 in terms of plaquemorphology, cytopathogenicity, growth kinetics, protein expression, andRNA production. Furthermore, analyses of the 3′ end sequence did notreveal an extra three (CGA) or four (CTAG) nucleotides ofvirus-unrelated sequence at the 3′ end of the viral RNA genomes derivedfrom any of the synthetic viruses. These results validate the use ofinfectious JEV cDNA clones developed in the present invention for futuremolecular genetics.

Example 7 Check the Possibility that the Transfected Cultures wereContaminated with the Parental Virus

While the above results strongly suggest that the JEV cDNA clones canproduce highly infectious RNA transcripts after SP6 or T7 polymerasetranscription, the possibility that the transfected cultures werecontaminated with the parental virus CNU/LP2 was not formally excluded.To assess this remote possibility, the present inventors used PCR-basedsite-directed mutagenesis to introduce a genetic marker (gm) into thepBAC^(SP6)/JVFLx/XbaI construct. Particularly, the point mutationA⁸¹⁷¹→C (silent) was placed inside the NS5 gene in PBAC SP6/JVFLx/XbaIby PCR-based site-directed mutagenesis to generatepBAC^(SP6)/JVFLx/gm/XbaI (FIG. 6A). The point mutation resulted in theacquisition of a unique Xho I restriction endonuclease recognition site.A fragment from pBAC^(SP6)/JVFLx/XbaI was first generated by PCR withprimer J48 represented by SEQ. ID. No 29, in which the Xho I was createdby the A⁸¹⁷¹→C substitution, and primer J3 represented by SEQ. ID. No30. The 665-bp Mlu I-Apa I fragment of the resulting amplicons was thenligated with the 4,802-bp Apa I-BsrG I and the 5,874-bp BsrG I-Mlu Ifragments of pBAC^(SP6)/JVFLx/XbaI, resulting in thepBAC^(SP6)/JVFLx/gm/XbaI construct. BHK-21 cells transfected with RNAtranscripts from Xba I-linearized MBN-treatedpBAC^(SP6)/JVFLx/gm/XbaI^(MBN) produced infectious virus containing thegenetic marker (denoted JVFLx/gm/XbaI^(MBN)) (FIG. 6A). The phenotypiccharacteristics of JVFLx/gm/XbaI^(MBN) did not differ from those of theoriginal virus JVFLx/XbaI^(MBN), indicating that the A⁸¹⁷¹→Csubstitution did not affect viral replication.

To verify that the JVFLx/gm/XbaI^(MBN) virus had been recovered from thecDNA template of pBAC^(SP6)/JVFLx/gm/XbaI^(MBN), the present inventorsserially passaged the recovered virus in BHK-21 cells at an MOI of 0.1.The viruses resulted from each passage were incubated with RNase A andDNase I to avoid the carryover of the input transcript RNA and templateplasmid cDNA (Mendez et al., J. Virol., 1998, 72, 4737-4745). Viral RNAsextracted from the JVFLx/gm/XbaI^(MBN) and JVFLx/XbaI^(MBN) virusesreleased at passages 1 and 3 were used in RT-PCR to amplify a 2,580-bpproduct that encompassed the A⁸¹⁷¹→C substitution (FIG. 6B, lanes 1, 3,and 5). Digestion of the amplified product from JVFLx/gm/XbaI^(MBN) withXho I resulted in two fragments of 1,506 and 1,074 bp (FIG. 6B, lanes 2and 4). On the other hand, the JVFLx/XbaI^(MBN)-derived RT-PCR productdid not digest with Xho I (FIG. 6B, compared lane 5 with lane 6),demonstrating that the A⁸¹⁷¹→C substitution was indeed present in theJVFLx/gm/XbaI^(MBN) virus. Thus, it was confirmed that the recoveredvirus JVFLx/gm/XbaI^(MBN) originated from the full-length infectiouscDNA pBAC^(SP6)/JVFLx/gm/XbaI^(MBN).

Example 8 Genetic Stability of Full-length Infectious JEV cDNA

A previous study has shown that constructs containing full-length JEVcDNA frequently acquired stabilizing nonsense mutations in the regionsencoding the structural proteins prM and E (Sumiyoshi et al., J. Virol.,1992, 66, 5425-5431). Since studies into the molecular genetics of JEVwill indispensably require a reliable infectious JEV molecular clone formanipulation, the present inventors manipulated pBAC^(SP6)/pJVFLx/XbaIin several ways and extensively investigated its genetic structure andfunctional integrity.

Particularly, the genetic structure and functional integrity of theinfectious JEV cDNAs were analyzed as follows. E. coli strain DH10B wastransformed with pBAC^(SP6)/JVFLx/XbaI, and two independently derivedclones were grown at 37° C. overnight in 10 Ml of 2×YT containing 12.5μg/Ml of chloramphenicol. Cells from these primary cultures weremaintained for 9 days by diluting them 10⁶-fold every day (Almazan etal., Proc. Natl. Acad. Sci. USA, 2000, 97, 5516-5521). In theexperimental conditions of the present invention, each passagerepresented approximately 20 generations, which was consistent withobservations made previously (Alamzan et al., Proc. Natl. Acad. Sci.USA, 2000, 97, 5516-5521). After the third, sixth, and ninth passages,large-scale preparation of the infectious cDNA plasmid was made by theSDS-alkaline method and purified further by cesium chloride densitygradient centrifugation (Sambrook et al., Molecular cloning, 1989, ColdSpring Harbor Laboratory). The genetic structure of the plasmid DNA wasmonitored by its restriction endonuclease pattern. The plasmidsextracted from the two cultures at passage 0, 3, 6 and 9 were examinedby restriction enzyme analysis. The restriction enzyme patterns atpassages 3, 6 and 9 did not differ visibly from those at passage 0.Thus, within the resolution of agarose gel electrophoresis analysis, thetwo infectious cDNA clones appeared to be structurally stable.

The functional integrity of the JEV cDNA plasmid was also investigatedby measuring the specific infectivities of the synthetic RNAstranscribed from the cDNA template, which was linearized by Xba Idigestion and MBN treatment. As a result, the infectivity of the RNAtranscripts made from the two cDNA clones did not differ between passage0 and passage 9 (FIG. 7). From the above result, it was confirmed thatthe infectious JEV cDNA remained functionally stable during serialgrowth in E. coli.

Example 9 Infectious JEV cDNA as a Vector for Foreign Gene Expression

As previously described (Burke and Monath, Flaviviruses, 2001,1043-1125, Lippincott Williams & Wilkins Publishers), the presentinventors found that JEV was able to replicate in a wide variety ofeukaryotic cells originating from a number of species, including humans,mice, monkeys, swine, dogs, cats, and hamsters. This suggests that JEVcould be useful as a vector for the expression of heterologous genes ina variety of different cells. To test this, two commonly used reportergenes, the Aequeorea victoria GFP and the Photinus pyralis LUC, wereinserted at the beginning of the viral 3′NTR of pBAC^(SP6)/JVFLx/XbaI asexpression cassettes driven by the IRES element of EMCV (FIG. 8A).

To create the pBAC^(SP6)/JVFLx/GFP/XbaI construct (FIG. 8A), a fragmentfrom pBAC^(SP6)/JVFLx/XbaI was amplified by PCR with the primer J72represented by SEQ. ID. No 31 and the primer J73 represented by SEQ. ID.No 32. A fragment was also amplified from pRSGFP-C1 with the primer J74represented by SEQ. ID. No 33 and the primer J75 represented by SEQ. ID.No 34. These two fragments were fused by the second round of PCR withthe primers J72 and J75. The 913-bp Kpn I-Nsi I fragment of theresulting amplicons was then ligated with the 8,011-bp Nsi I-Pac 1 and11,021-bp Pac I-Kpn I fragments of pBAC^(SP6)/JVFLx/XbaI, resulting inthe pBAC^(SP6)/JVFLx/GFP/XbaI construct.

To generate the pBAC^(SP6)/JVFLx/LUC/XbaI construct (FIG. 8A), afragment of pBAC^(SP6)/JVFLx/XbaI was amplified with the primer J72 andthe primer J76 represented by SEQ. ID. No 35. A fragment was alsoamplified from pACNR/NADLcIn-/LUC (provided by Dr. Charles M. Rice) withthe primer J77 represented by SEQ. ID. No 36 and the primer J78represented by SEQ. ID. No 37. These two fragments were then fused bythe second round of PCR with the primers J72 and J78. The 1,801-bp KpnI-Nsi I fragment of the resulting amplicons was then ligated with the8,011-bp Nsi I-Pac 1 and 11,021-bp Pac I-Kpn I fragments ofpBAC^(SP6)/JVFLx/XbaI, leading to pBAC^(SP6)/JVFLx/LUC/XbaI.

To generate pBAC^(SP6)/JVFLx/LUC^(REP-)/XbaI (FIG. 8A), which containsan 83-nucleotide deletion (nt 5,581 to 5,663) in the middle of the NS3gene that results in premature termination of viral translation at nt5,596, a fragment of pBAC^(SP6)/JVFLx/LUC/XbaI was amplified with theprimer J89 represented by SEQ. ID. No 38 and the primer J91 representedby SEQ. ID. No 39. A fragment was also amplified from pBACSP6/JVFLx/LUC/XbaI with the primer J92 represented by SEQ. ID. No 40 andthe primer J93 represented by SEQ. ID. No 41. These two fragments werethen fused by the second round of PCR with the primers J89 and J93. The3,960-bp Sfi I-Eag I fragment of the resulting amplicons was thenligated with the 6,493-bp Eag I-Sfi I and 10,297-bp Sfi I-Sfi Ifragments of pBAC^(SP6)/JVFLx/LUC/XbaI, leading topBAC^(SP6)/JVFLx/LUC^(REP-)/XbaI.

A deletion of 9 to 25 nucleotides exists at the beginning of the viral3′NTR in CNP/LP2 and three other fully sequenced JEV strains (Williamset al., J. Gen. Virol., 2000, 81, 2471-2480; Nam et al., Am. J. Trop.Med. Hyg., 2001, 65, 388-392; Jan et al., Am. J. Troop. Med. Hyg., 1996,55, 603-609), suggesting that this may be a good site to insert theforeign genes. Therefore, when BHK-21 cells were transfected with thesynthetic RNAs transcribed from pBAC^(SP6)/JVFLx/GFP/XbaI andpBAC^(SP6)/JVFLx/LUC/XbaI cDNAs, the insertion did not alter thespecific infectivity of the synthetic RNA transcripts.

To examine GFP expression, naïve BHK-21 cells were transfected withinfectious synthetic RNA transcribed from thepBAC^(SP6)/JVFLx/GFP/XbaI^(MBN) template and examined by confocalmicroscopy. Particularly, BHK-21 cells were mock-transfected ortransfected with 2 μg of JVFLx/GFP/XbaI^(MBN) RNA. Transfected cells(1×10⁵) were incubated for 30 hr in a four-well chamber slide. Cellswere washed twice with PBS, fixed by incubation for 30 min at 25° C. inPBS containing 0.37% (v/v) formaldehyde, and mounted with 0.2 Ml of 80%glycerol. Cells were viewed by confocal microscopy and analyzed. As aresult, BHK-21 cells expressing GFP displayed green fluorescence in boththe nucleus and the cytoplasm (FIG. 8B, JVFLx/GFP/XbaI^(MBN)) becauseGFP is small enough to permit diffusion between the nucleus and thecytoplasm. As expected, this fluorescence was not observed inmock-transfected cells (FIG. 8B, mock) or in cells transfected with RNAtranscripts from pBAC^(SP6)/JVFLx/XbaI^(MBN).

To monitor the induction of LUC over time in a quantitative manner, thepresent inventors produced not only replication-competent RNAtranscripts from pBAC^(SP6)/JVFLx/LUC/XbaI^(MBN) but alsoreplication-incompetent RNA transcripts frompBAC^(SP6)/JVFLx/LUC^(REP-)/XbaI^(MBN) (FIG. 8A). ThepBAC^(SP6)/JVFLx/LUC^(REP-)/XbaI^(MBN) template contains an83-nucleotide deletion (nt 5,581 to nt 5,663) in the middle of the NS3gene that prematurely terminates viral translation at nt 5,596 (see * inFIG. 8A, pBAC^(SP6)/JVFLx/LUC^(REP-)/XbaI^(MBN)).

For the LUC assay, BHK-21 cells (8×10⁶) were mock-transfected ortransfected with 2 μg of JVFLx/LUC/XbaI^(MBN) RNA orJVFLx/LUC^(REP-)/XbaI^(MBN) RNA. Cells were seeded at a concentration of6×10⁵ cells/well in a six-well plate and cultivated. At the given timepoints, the cells were washed with Ca²⁺- and Mg²⁺-free PBS solution andthen lysed by adding 0.2 W of lysis buffer [25 mM Tris-phosphate (pH7.8), 2 mM DTT, 2 mM 1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid,10% glycerol, 1% Triton X-100(v/v)] to each well. Cell lysates wereincubated for 10 min at room temperature, and cellular debris was thenremoved by centrifugation. The supernatants were quickly placed at −80°C. for storage until use. To determine the LUC activity, 20 μl of thecell lysates was placed in a luminometer tube containing 100 μl of LUCassay reagent [20 mM Tricine, 1.07 mM (MgCO₃)₄Mg(OH)₂.5H₂O, 2.67 mMMgSO₄, 0.1 mM EDTA, 33.3 mM DTT, 270 μM coenzyme A, 470 μM luciferin(Promega), 530 μM ATP]. The activity was usually measured for 10 sec.Each data point represents the results of three independent experiments.

As a result, in BHK-21 cells transfected with the replication-competentJVFLx/LUC/XbaI^(MBN) RNA (FIG. 8C, ●), the initial LUC activity 6 hrposttransfection was 2.4×10³+0.2×10³ relative light units (RLU). Thisactivity was dramatically increased to 5.3×10⁴±0.1×10⁴ RLU 30 hrposttransfection and reached 7.8×10⁵±0.6×10⁵ RLU 54 hr posttransfection,at which point >50% of the cells were dying. In contrast, in BHK-21cells transfected with the replication-incompetentJVFLx/LUC^(REP-)/XbaI^(MBN) RNA, the initial LUC activity 6 hrposttransfection was 1.9×10³+0.4×10³ RLU (FIG. 8C, ∘), like theJVFLx/LUC/XbaI^(MBN)-transfected cells (FIG. 8C, ●), but this activitygradually decreased over time to 16±1.2 RLU at 54 posttransfection,which is at the level of background luminescence of naïve cells (FIG.8C, ∘) Thus, the level of LUC activity expressed over time varieddepending on the presence or absence of viral replication.

The present inventors produced full-length infectious recombinant JEVcDNAs having GFP and LUC genes according to the method explainedhereinbefore. BHK-21 cells were transfected with JEV RNA transcriptstranscribed from the recombinant JEV cDNAs, and then, recombinant JEVJVFLx/GFP/XbaI^(MBN) and JVFLx/LUC/XbaI^(MBN) containing GFP and LUCgenes were recovered from culture supernatants. The expression of GFPand LUC genes in the recombinant JEV was investigated after infecting avariety of animal cell lines (BHK-21, Vero, NIH/3T3, ST, HeLa, MDCK,CRFK, B103, and SHSY-5Y), which have been generally used in the field ofbiology and medicine, with the virus. As a result, GFP or LUC geneinserted in virus genome was expressed in all cells tested (Table 4).Thus, it was confirmed that recombinant JEV cDNAs, JEV RNA transcripts,and recombinant JEV viral particles could be effectively used as avector for expression of foreign heterologous genes in a variety of celltypes.

TABLE 4 Expression of GFP and LUC genes engineered in the infectious JEVcDNAs Cell line GFP expression^(a) LUC induction^(b) BHK-21 ExpressedExpressed Vero Expressed Expressed HeLa Expressed Expressed MDCKExpressed Expressed CRFK Expressed Expressed NIH/3T3 Expressed ExpressedST Expressed Expressed B103 Expressed Expressed SHSY-5Y ExpressedExpressed ^(a)Expression of GFP protein was analyzed after infectingcells with recombinant JEV JVFLx/GFP/XbaI^(MBN). ^(b)Expression of LUCprotein was analyzed after infecting cells with recombinant JEVJVFLx/LUC/XbaI^(MBN).

Example 10 Utility of the Infectious JEV cDNA for a Novel HeterologousGene Expression System

The present inventors further investigated the utility of JEV-basedexpression system in expressing foreign genes of interest. First, thepresent inventors engineered the full-length viral genome to expressthree commonly used and variously sized heterologous reporter genes,namely, an improved version of the Aequorea victoria GFP gene (EGFP, 768bp), the LUC gene from Photinus pyralis (1653 bp), and the LacZ (3012bp) gene (FIG. 9B). The present inventors also introduced the dominantselective marker PAC (600 bp), which facilitates resistance to the drugpuromycin (FIG. 9B).

<10-1> Construction and Characterization of Heterologous Gene-encodingInfectious Recombinant JEVs That are Based on the BicistronicFull-Length Infectious JEV cDNA that Serves as a BAC.

<10-1-1> Plasmid Construction of Infectious Recombinant JEV Vectors

All plasmids were constructed by standard molecular biology protocols(Sambrook et al., Molecular cloning: a laboratory manual, 2^(nd) ed.Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989) and allregions amplified by PCR were verified by sequencing. All of therecombinant JEV vectors used in the present invention were constructedbased on pBAC^(SP6)/JVFLx/XbaI (Yun et al., J. Virol., 2003, 77,6450-6465), which is designated as pJEV/FL hereinafter (FIG. 9A).

The present inventors constructed a set of four infectious recombinantJEV vectors expressing the LUC, EGFP, LacZ, and PAC genes. pJEV/FL/LUCis identical to the construction designated as pBAC^(SP6)/JVFLx/LUC/XbaIhereinbefore in the Example 9 (FIG. 9B). To construct pJEV/FL/LacZ, the2,409-bp Kpn I-Avr II fragment of pJEV/FL/LUC was first subcloned intopGEM3Zf(+) which was digested with Kpn I and Xba I, resulting inpGEM/LUC. The 3,177-bp Nco I-Stu I fragment of pSinRep3/LacZ (a generousgift from Dr. Charles Rice) was ligated to the 3,935-bp Nco I-Nsi I (T4DNA polymerase-treated) fragment of pGEM/LUC, leading to pGEM/LacZ. The3,873-bp Kpn I-Not I fragment of pGEM/LacZ was ligated to the 7,456-bpNot I-Pac 1 and 11,021-bp Pac I-Kpn I fragments of pJEV/FL/LUC, creatingpJEV/FL/LacZ (FIG. 9B). To facilitate the construction of pJEV/FL/EGFP,the 5,792-bp Sac II-Not I fragment of pJEV/FL/LacZ was inserted intopRS2, which was digested with the same enzymes, resulting in pRS/LacZ. Afragment of the sequence coding for EGFP was produced by PCRamplification of pEGFP-C1 with the primers EGFPF (represented by SEQ.ID. No 49) and EGFPR (represented by SEQ. ID. No 50). The 773-bp NcoI-Stu I portion of the EGFP fragment amplicons was ligated to the3,241-bp EcoR V-Sac II and 2,062-bp Sac II-Nco I fragments of pRS/LacZ,resulting in pRS/EGFP. The 3,406-bp Sac II-Not I fragment of pRS/EGFPwas ligated to the 7,456-bp Not I-Pac 1 and 9,102-bp Pac I-Sac IIfragments of pJEV/FL/LUC, leading to pJEV/FL/EGFP (FIG. 9B). To generatepJEV/FL/PAC, a fragment of pACNR/NADLcIns⁻/PAC (a generous gift from Dr.Charles Rice) was PCR-amplified with primers PACF (represented by SEQ.ID. No 51) and PACR (represented by SEQ. ID. No 52). The 881-bp DraIII-Nsi I portion of the resulting amplicons was ligated to the 8,011-bpNsi I-Pac I, 10,096-bp Pac I-Nde I, and 842-bp Nde I-Dra III fragmentsof pJEV/FL/LUC, resulting in pJEV/FL/PAC (FIG. 9B). An expressioncassette driven by the EMCV IRES was inserted at the beginning of theviral 3′ NTR of pJEV/FL (FIGS. 9A and 9B).

<10-1-2> Assay for EGFP Expression

Cells were seeded in a four-well chamber slide for 36-48 hrposttransfection. After incubation, cells were fixed by being incubatedin PBS containing 0.37% (v/v) formaldehyde and then mounted with 0.2 ml80% glycerol. Cells were observed under a confocal microscope outfittedwith an appropriate filter. The expression of EGFP was also examined byflow cytometric analysis. Particularly, the cells were trypsinized,washed once with PBS, and resuspended in 0.37% (v/v) formaldehyde inPBS, followed by analysis with a FACScan flow cytometer FACSCalibur(Becton Dickinson). Dead cells were excluded by appropriate forward andside light-scattering gates. Ten thousand viable cells were counted.

<10-1-3> β-Galactosidase Assay

Cells were washed once with PBS, fixed with 0.05% (v/v) glutaraldehydein PBS for 15 min at room temperature, and carefully washed three timeswith PBS. The cells were assessed for β-gal activity by being incubatedin staining solution [5 mM potassium ferricyanide, 5 mM potassiumferrocyanide, 2 mM MgCl₂ in PBS] with5-bromo-4-chloro-3-indolyl-β-galactopyranoside (Sigma) at 37° C.

<10-1-4> Luciferase Assay

Cells were analyzed for LUC activity by using the substrate luciferin(Promega) as described hereinbefore (Yun et al., J. Virol., 2003, 77,6450-6465). Each experiment was performed in triplicate and the meanvalues are presented.

<10-1-5> Puromycin Selection

Cells were seeded in 6-well plates at 37° C. for 6 hr. To measurePur^(R) foci formation, the cells were overlaid with 0.5% SeaKem LEagarose (FMC BioProducts, Rockland, Me.) in MEM containing 10%heat-inactivated FBS and penicillin/streptomycin and incubated at 37° C.for 2 days. Thereafter, the plates were incubated for an additional 3days in the absence or presence of puromycin (10 μg/ml). After theselection, the Pur^(R) foci were visualized by crystal violet stainingof the formaldehyde-fixed cells (Yun et al., J. Virol., 2003, 77,6450-6465). For Pur^(R) cell culture, the cells were left unplugged withthe agarose and incubated in complete medium at 37° C. for 2 days.Subsequently, the cells were cultivated in complete media containing 10μg/ml puromycin and 24-48 hr after selection, the surviving cells werevisualized by staining with crystal violet.

<10-1-6> Heterologous Proteins are Expressed in BHK-21 CellsTransfected/Infected with Recombinant Synthetic JEV RNAs/VirusesContaining an Additional Expression Unit

To examine whether the insertion of the expression cassette altered itsspecific infectivity/replication, the present inventors examined thespecific in vitro infectivity of the synthetic RNAs that had beentranscribed from the four SP6-driven foreign gene-bearing infectious JEVcDNA constructs (Table 5). Purified pJEV/FL and its derivative plasmidswere linearized by digestion with Xba I followed by treatment with MBN.The linearized plasmids were used in vitro transcription reactions (25μl) employing SP6 RNA polymerase, as described hereinbefore. Aftertranscription, the reaction mixtures were further incubated with 10 UDNase I for 30 min and extracted with phenol-chloroform-isoamylalcohol.RNA yields were quantified on the basis of [³H]UTP incorporation asmeasured by RNA absorption to DE-81 filter paper (Whatman, Maidstone,UK). RNA (2 μg) was transfected into cells by electroporation asdescribed hereinbefore (Yun et al., J. Virol., 2003, 77, 6450-6465).

The synthetic RNAs derived from pJEV/FL/PAC, pJEV/FL/EGFP, pJEV/FL/LUC,and pJEV/FL/LacZ introduced into susceptible BHK-21 cells had specificinfectivities of 3.5×10⁶, 2.5×10⁶, 3.4×10⁶, and 1.1×10⁶ PFU/μg,respectively, which are similar to the infectivity of the parentalpJEV/FL (3.2×10⁶ PFU/μg). However, the BHK-21 cells transfected with therecombinant synthetic RNAs did form homogeneous smaller plaques than thepJEV/FL-transfected cells (FIG. 9C). This accords with the delayedproduction of infectious virus (Table 5, Virus titer) and reducedcytopathogenicity (Table 5, CPE) that was observed in the recombinantRNA-transfected cells. The present inventors also showed that thedelayed accumulation of the viral proteins (FIG. 9D) in the recombinantRNA-transfected BHK-21 cells correlated with the length of foreign genethat had been inserted (FIG. 9B).

TABLE 5 Specific infectivity of in vitro RNA transcripts generated fromfull-length JEV cDNA derivatives containing various reporter genes andrecombinant virus titer Template used Virus titer^(c) forInfectivity^(b) (PFU/ml) transcription^(a) (PFU/μg of RNA) 48 hr 72 hrCPE^(d) pJEV/FL 3.2 × 10⁶ 3.0 × 10⁶ 5.1 × 10⁵ ++++ pJEV/FL/PAC 3.5 × 10⁶6.2 × 10⁴ 4.0 × 10⁵ ++ pJEV/FL/EGFP 2.5 × 10⁶ 9.0 × 10⁴ 2.1 × 10⁵ ++pJEV/FL/LUC 3.4 × 10⁶ 2.0 × 10⁴ 3.2 × 10⁵ + pJEV/FL/LUC^(REP-) 0 0 0 −pJEV/FL/LacZ 1.1 × 10⁶ 1.1 × 10⁴ 1.3 × 10⁵ + ^(a)All JEV cDNA templatesused for in vitro transcription reaction were prepared by linearizationwith Xba I digestion, which was followed by treatment with MBN.^(b)After in vitro transcription with SP6 RNA polymerase, samples wereused to electroporate BHK-21 cells, and infectious plaque centers weredetermined (Yun et al., J. Virol., 2003, 77, 6450-6465). ^(c)Virustiters at 48 hr and 72 hr postelectroporation. ^(d)Virus-induced CPE wasobserved after electroporation with RNA transcripts generated fromfull-length JEV cDNA derivatives. At 24 hr postelectroporation, strongCPE was observed for the parental pJEV/FL as indicated by ++++. ForpJEV/FL/PAC and pJEV/FL/EGFP, CPE was observed at 60 hrpostelectroporation as indicated by ++. For pJEV/FL/LacZ, clear CPEbegan to be displayed at 72 hr postelectroporation as indicated by +. −indicates no CPE.

EGFP, LUC, LacZ and PAC expression using infectious JEV cDNA is shown inFIG. 10. The JEV/FL/EGFP RNA-transfected BHK-21 cells showed brightgreen fluorescence under a fluorescence microscope (FIG. 10A). The greenfluorescent cells (−), as determined by flow cytometry analysis,comprised 99.7% of the cells compared to mock-transfected cells (. . .)(FIG. 10B). FIG. 10C demonstrates the X-gal staining pattern of theJEV/FL/LacZ RNA-expressing BHK-21 cells. The present inventors alsomonitored the LUC activity over time of BHK-21 cells that had beentransfected with either the replication-competent JEV/FL/LUC RNA (●) orthe replication-incompetent JEV/FL/LUC^(REP-) RNA (∘), which lacks asection that prematurely terminates viral translation at nt 5596 (*).This demonstrated that the increased viral replication correlated withincreased LUC activity (FIG. 10D), as previously described (Yun et al.,J. Virol., 2003, 77, 6450-6465). Furthermore, selection of theJEV/FL/PAC RNA-transfected BHK-21 cells with puromycin (FIG. 10E)revealed the JEV/FL/PAC RNA-transfected cells survived and becomeconfluent in the puromycin-containing media (dish 7) or formed Pur^(R)foci under semisolid agar overlaid with puromycin-containing media (dish8), whereas the mock-transfected cells died within 24 hr of selection(dishes 5-6). As expected, both cell types became confluent in theabsence of puromycin (dishes 1-4).

<10-2> Construction and Vector Characteristics of JEV Viral Replicons

<10-2-1> Plasmid Construction of JEV Viral Replicon Vectors

Plasmids for all JEV viral replicons were constructed based onpJEV/FL/LUC by engineering in-frame deletions in the coding sequences ofthe structural proteins. All deletions were distinguished by a novel XhoI site that resulted in the insertion of two residues, namely, Leu andGlu. First, the present inventors generated a set of four JEV viralreplicon vectors containing a single in-frame deletion in eachstructural protein. To construct pJEV/Rep/ΔCC/LUC, which contains a273-nucleotide deletion (nt 132-404) in the C gene, two fragments weresynthesized by PCR amplification of pJEV/FL, namely, fragment C1 withprimers DelF (represented by SEQ. ID. No 53) and C1R (represented bySEQ. ID. No 54), and fragment C2 with primers C2F (represented by SEQ.ID. No 55) and DelR (represented by SEQ. ID. No 56). Two fragments (the267-bp Pac I-Xho I portion of the C1 fragment amplicons and the 226-bpXho I-BsiW I portion of the C2 fragment amplicons) were ligated to the20,073-bp BsiW I-Pac I fragment of pJEV/FL/LUC, resulting in thepJEV/Rep/ΔCC/LUC construct. To generate pJEV/Rep/ΔC/LUC, which containsa 204-nucleotide deletion (nt 201-404) in the C gene, fragment C3 frompJEV/FL was amplified by PCR with the primers DelF and C3R (representedby SEQ. ID. No 57). The 336-bp Pac I-Xho I fragment of the resultingamplicons was ligated to the 12,850-bp Xho I-Rsr II and 7,449-bp RsrII-Pac I fragments of pJEV/Rep/ΔCC/LUC, resulting in the pJEV/Rep/ΔC/LUCconstruct. To create pJEV/Rep/ΔprM/LUC, which contains a 282-nucleotidedeletion (nt 531-812) in the prM gene, two fragments were obtained bythe PCR amplification of pJEV/FL, namely, fragment prM1 with the primersDelF and prM1R (represented by SEQ. ID. No 58), and fragment prM2 withprimers prM2F (represented by SEQ. ID. No 59) and DelR. Two fragments(the 666-bp Pac I-Xho I portion of the prM1 fragment amplicons and the1,616-bp Xho I-Sfi I portion of the prM2 fragment amplicons) wereligated to the 10,264-bp Sfi I-Nsi 1 and 8,011-bp Nsi I-Pac I fragmentsof pJEV/FL/LUC, resulting in the pJEV/Rep/ΔprM/LUC construct. Toengineer pJEV/Rep/ΔE/LUC, which contains a 1,170-nucleotide deletion (nt1,032-2,201) in the E gene, two fragments were produced by PCRamplification of pJEV/FL, namely, fragment E1 with primers DelF and E1R(represented by SEQ. ID. No 60), and fragment E2 with primers E2F(represented by SEQ. ID. No 61) and DelR. Two fragments (the 1,167-bpPac I-Xho I portion of the prM1 fragment amplicons and the 227-bp XhoI-Sfi I portion of the prM2 fragment amplicons) were ligated to the10,264-bp Sfi I-Nsi 1 and 8,011-bp Nsi I-Pac I fragments of pJEV/FL/LUC,resulting in the pJEV/Rep/ΔE/LUC construct (FIG. 11A).

Second, the present inventors constructed a panel of three JEV viralreplicon vectors that contain a double in-frame deletion in the JEVstructural genes. Two fragments of pJEV/FL/LUC (the 10,264-bp Sfi I-Nsi1 and 8,011-bp Nsi I-Pac I fragments) were ligated to either (i) the438-bp Pac I-Hind III fragment of pJEV/Rep/ΔC/LUC and the 1,646-bp HindIII-Sfi I fragment of pJEV/Rep/ΔprM/LUC to generatepJEV/Rep/ΔC+ΔprM/LUC, (ii) the 866-bp Pac I-Mlu I fragment ofpJEV/Rep/ΔC/LUC and the 330-bp Mlu I-Sfi I fragment of pJEV/Rep/ΔE/LUCto generate pJEV/Rep/ΔC+ΔE/LUC, or (iii) the 788-bp Pac I-Mlu I fragmentof pJEV/Rep/ΔprM/LUC and the 330-bp Mlu I-Sfi I fragment ofpJEV/Rep/ΔE/LUC to generate pJEV/Rep/ΔprM+ΔE/LUC (FIG. 11A).

Third, the present inventors created a set of two JEV viral repliconvectors in which all JEV structural proteins were lacking. To generatepJEV/Rep/ΔC+ΔprM+ΔE/LUC, two fragments of pJEV/FL/LUC (the 10,264-bp SfiI-Nsi 1 and 8,011-bp Nsi I-Pac I fragments) were ligated to the 590-bpPac I-Mlu I fragment of pJEV/Rep/ΔC+ΔprM/LUC and the 330-bp Mlu I-Sfi Ifragment of pJEV/Rep/ΔE/LUC. The present inventors also constructedpJEV/Rep/NS1/LUC, which contains the 35 N-terminal and 24 C-terminalamino acids of the C protein followed immediately by the N-terminus ofthe NS1 protein and the rest of the viral genome. A fragment frompJEV/Rep/ΔC/LUC was first synthesized by PCR with the primers DelF andNS1R (represented by SEQ. ID. No 62). A fragment from pJEV/FL was thensynthesized with the primers NS1F (represented by SEQ. ID. No 63) and RR(represented by SEQ. ID. No 64). These two fragments were fused by asecond round of PCR with the primers DelF and RR. The 474-bp Pac I-ApaLI fragment of the resulting amplicons was ligated to the 3,038-bp ApaLI-BamH 1 and 15,122-bp BamH I-Pac I fragments of pJEV/FL/LUC, leading topJEV/Rep/NS1/LUC (FIG. 11A).

In addition to pJEV/Rep/ΔC+ΔprM+ΔE/LUC and pJEV/Rep/NS1/LUC, the presentinventors also constructed eight other JEV viral replicon vectors. The6,797-bp BamH I-Not I fragment of pJEV/FL/EGFP was ligated to either (i)the 11,529-bp BamH I-Not I fragment of pJEV/Rep/ΔC+ΔprM+ΔE/LUC to createpJEV/Rep/ΔC+ΔprM+ΔE/EGFP, or (ii) the 10,968-bp BamH I-Not I fragment ofpJEV/Rep/NS1/LUC to create pJEV/Rep/NS1/EGFP. The 5,792-bp Sac II-Not Ifragment of pJEV/FL/LacZ was ligated to either (i) the 7,456-bp NotI-Pac I and the 7,464-bp Pac I-Sac II fragments ofpJEV/Rep/ΔC+ΔprM+ΔE/LUC to create pJEV/Rep/ΔC+ΔprM+ΔE/LacZ, or (ii) the7,456-bp Not I-Pac I and the 6,903-bp Pac I-Sac II fragments ofpJEV/Rep/NS1/LUC to create pJEV/Rep/NS1/LacZ. The 6,663-bp BamH I-Not Ifragment of pJEV/FL/PAC was ligated to either (i) the 11,529-bp BamHI-Not I fragment of pJEV/Rep/ΔC+ΔprM+ΔE/LUC to createpJEV/Rep/ΔC+ΔprM+ΔE/PAC, or (ii) the 10,968-bp BamH I-Not I fragment ofpJEV/Rep/NS1/LUC to create pJEV/Rep/NS1/PAC.

<10-2-2> Heterologous Proteins are Expressed From a Variety ofSelf-replicating Self-limiting JEV Viral Replicons

To independently express foreign genes using the JEV RNA replicationmachinery, the present inventors generated a panel of self-replicatingself-limiting viral replicons that meet stringent safety concerns (FIG.11A). Initially, the present inventors used the LUC reporter as theheterologous gene as it facilitates the monitoring of viral replicationin a sensitive and quantitative manner. Thus, a variety of repliconvectors were carefully engineered in the context of pJEV/FL/LUC by thein-frame deletion of one, two, or all of the viral structural genes (C,prM, and E), in consideration with the membrane orientation of eachprotein (FIG. 11A).

The LUC activities of the BHK-21 cells that had been transfected withthe various viral replicons were plotted over time (FIG. 11B). In BHK-21cells transfected with the replication-competent JEV/FL/LUC RNA (●,black) as a positive control, the initial LUC activity at 6 hrposttransfection was 5.5±0.3×10³ RLU. This activity dramaticallyincreased to 2.7±0.5×10⁶ RLU at 48 hr posttransfection and wasmaintained through to 96 hr posttransfection. In BHK-21 cellstransfected with the replication-incompetent JEV/FL/LUC^(REP-) RNA (♦,black), the initial LUC activity expressed from the input viral RNA at 6hr posttransfection was similar, namely, 5.2±0.6×10³ RLU. However, thisactivity gradually decreased over time to 8.8±1.0 RLU at 96 hrposttransfection, which is equivalent to the background luminescence ofnaïve cells. Apart from pJEV/Rep/ΔCC/LUC (♦, blue), which lacks asequence that is complementary to a proposed cyclization sequence in the3′NTR that is conserved in all flaviviruses (Bredenbeek et al., J. Gen.Virol., 2003, 84, 1261-1268; Lo et al., J. Virol., 2003, 77,10004-10014; Khromykh et al., J. Virol., 2001, 75, 6719-6728), the LUCactivities of the BHK-21 cells transfected with the viral repliconslacking part of one or more structural protein genes were almostidentical in the 6-48 hrs posttransfection to those of thereplication-competent JEV/FL/LUC RNA-transfected BHK-21 cells (●,black). Thereafter, however, these activities decreased dramaticallyover time due to a lack of viral spread, similar to JEV/FL/LUC^(REP-).Interestingly, the LUC activities due to JEV/Rep/NS1/LUC RNA (●, green),but not to JEV/Rep/ΔC+ΔprM+ΔE/LUC RNA (▪, green), were approximately5-fold higher at all time points compared to the activities of the otherreplication-competent viral replicons.

The LUC expression profiles agreed with the viral protein accumulation(FIG. 1C), as quantified by immunoblotting with JEV-specific hyperimmunesera. The present inventors also confirmed that other reporter genescould be efficiently expressed in various commonly used animal cells byusing JEV-based replicon vectors such as pJEV/Rep/NS1 andpJEV/Rep/ΔC+ΔprM+ΔE.

<10-3> Construction of the Packaging System for JEV Viral Replicons

<10-3-1> Plasmid Construction of JEV Structural Protein ExpressionVectors Based on the pSinRep19 Vector

The present inventors constructed three JEV structural proteinexpression vectors based on pSinRep19 (Agapov et al., Proc. Natl. Acad.Sci. USA, 1998, 95, 12989-12994). For pSinRep19/JEV C-E, a fragment ofpJEV/FL was amplified with primer JEVCF (5′-GATTCTAGAATGACTAAAAAACCA,represented by SEQ. ID. No 65), which incorporates an Xba I site(underlined) and primer JEVER (5′-GATGTTTAAACTATTAAGCATGCACATTGGT,represented by SEQ. ID. No 66), which incorporates a Pme I site(underlined). The 2,393-bp Xba I-Pme I fragment of the resultingamplicons was ligated to the 10,864-bp Xba I-Mlu I (T4 DNApolymerase-treated) fragment of pSinRep19 to construct pSinRep19/JEV C-E(FIG. 12A). For pSinRep19/JEV C-NS1, a fragment was obtained by PCRamplification of pJEV/FL with primer JEVCF and primer JEVNS1R(5′-GATGTTTAAACTATTAAGCATCAACCTGTGA, represented by SEQ. ID. No 67),which incorporates a Pme I site (underlined). The 3,449-bp Xba I-Pme Ifragment of the resulting amplicons was then ligated to the 10,864-bpXba I-Mlu I (T4 DNA polymerase-treated) fragment of pSinRep19 toconstruct pSinRep19/JEV C-NS1 (FIG. 12A). For pSinRep19/JEV C-E-BglII,the 2,559-bp Xba I-Bgl II (T4 DNA polymerase-treated) fragment ofpSinRep19/JEV C-NS1 was ligated to the 10,864-bp Xba I-Mlu I (T4 DNApolymerase-treated) fragment of pSinRep19 (FIG. 12A).

<10-3-2> Generation of Packaging Cell Lines for JEV-derived RepliconVector RNAs.

The utility of the JEV replicon-based expression vectors was elaboratedby developing packaging cell lines (PCLs) that constitutively expressall the structural proteins of JEV (C, prM, and E) and allow thetrans-complementation of the efficient packaging of JEV viral replicons.Based on the pSinRep19 expression vector that contains the PAC genedriven by the subgenomic promoter, which facilitates selection (FIG.12A), the present inventors constructed three different JEV structuralprotein expression cassette constructs that encode the sequences for C-E(pSinRep19/JEV C-E), C-E plus the 58 N-terminal residues of NS1(pSinRep19/JEV C-E-BglII), and C-NS1 (pSinRep19/JEV C-NS1).

The protein expression yielded by these vectors was evaluated in BHK-21cells transfected with the synthetic RNAs that had been transcribed invitro from the corresponding vector. pSinRep19 and its derivatives werelinearized by digestion with Xho I. The linearized plasmids were used invitro transcription reactions (25 μl) employing SP6 RNA polymerase, asdescribed hereinbefore. After transcription, the reaction mixtures werefurther incubated with 10 U DNase I for 30 min and extracted withphenol-chloroform-isoamylalcohol. RNA yields were quantified on thebasis of [³H]UTP incorporation as measured by RNA absorption to DE-81filter paper (Whatman, Maidstone, UK). RNA (2 μg) was transfected intocells by electroporation as described hereinbefore (Yun et al., J.Virol., 2003, 77, 6450-6465). When the cell lysates from the transfectedcells were analyzed by immunoblotting with JEV-specific hyperimmunesera, equal amounts of viral glycoprotein E were detected in the BHK-21cells transfected with each of the three vectors (FIG. 12B). Asdesigned, the NS1 protein was detected only in the SinRep19/JEV C-NS1RNA-transfected cells (FIG. 12B).

Two approaches to produce JEV viral replicon particles (VRPs) areillustrated in FIG. 12C. One involves the transient cotransfection of invitro-transcribed JEV replicon vector RNA with the SinRep19 vector RNAthat expresses the JEV structural proteins. Titering and monitoring ofthe packaged VRPs was made possible by infecting naïve BHK-21 cells withthe VRPs and then assaying for reporter gene expression. Cotransfectionof SinRep19/JEV C-NS1 RNA with EGFP-expressing JEV viral replicon RNAs[either JEV/Rep/ΔC+ΔprM+ΔE/EGFP (□, green) or JEV/Rep/NS1/EGFP (▪,green)] in several experiments produced 1.1-4.3×10⁴ infectious units/ml(IU/ml) of VRPs (FIG. 12D). Similar results were obtained usingLacZ-expressing JEV viral replicons, namely, eitherJEV/Rep/ΔC+ΔprM+ΔE/LacZ (□, blue) or JEV/Rep/NS1/LacZ (▪, blue). Nodifference was observed when the SinRep19/JEV C-NS1 or the SinRep19/JEVC-E-BglII JEV structural protein expression cassettes were used.However, cotransfection of the SinRep19/JEV C-E vector RNA with theviral replicons expressing either EGFP or LacZ produced≈100-fold fewerVRPs (FIG. 12D). These observations were confirmed by cotransfecting allJEV structural protein expression vector RNAs with the LUC-expressingJEV replicon RNAs, namely, JEV/Rep/ΔC+ΔprM+ΔE/LUC (□, black) orJEV/Rep/NS1/LUC (●, black) (FIG. 12D)

The other approach to producing JEV VRPs is based on using a continuousPCL, which is established by transfecting cells with the JEV structuralprotein expression vector RNA and selecting with puromycin. The BHK-21cells were transfected with JEV structural protein expression vectorRNAs as mentioned hereinbefore. After transfection, the cells wereseeded for ≈24 hr and the media were replaced with fresh complete mediacontaining 10 μg/ml puromycin (Sigma). Thereafter, the cells weremaintained in the presence of puromycin and passaged or frozen as theparental BHK-21 cells.

The selected cells were shown to stably express the JEV structuralproteins without any deleterious effects to the host cell and wereslightly more efficient in producing JEV-based VRPs than the parentalBHK-21 cells. In all cases, higher VRP titers (1.0×10³-1.2×10⁵ IU/ml)were obtained upon transfection of these PCLs with the JEV viralreplicon vector RNAs, as compared to the protocol involving thecotransfection of the parental BHK-21 cells with two vector RNAs (FIG.12E).

To test for the presence of replication-competent viral particles in thepackaging system developed in the present invention, naïve BHK-21 cellswere infected with 3×10⁵ IU of the VRPs at an MOI of 1 for 72 hr. Theundiluted supernatant obtained from the infected cells was furtherpassaged three times to amplify the possible existence of very lowamounts of replication-competent viral particles. At the end of thesepassages, the infected cells were tested for the expression of thereporter gene or viral protein by IFA using JEV-specific hyperimmunesera. No replication-competent viral particles were ever detected.Furthermore, Sindbis replicon RNAs that express JEV structural proteinswere not encapsidated in the released VRPs.

INDUSTRIAL APPLICABILITY

As explained hereinbefore, the authentic nucleotide sequence of JEVgenomic RNA and the full-length infectious JEV cDNA of the presentinvention synthesized therefrom can be used not only for theidentification of the JEV genes, but also for the molecular biologicalstudies including JEV replication, transcription, and translation.Moreover, they can also be applied to the development of the therapeuticagents, vaccines, diagnostic reagents, and diagnostic devices forJapanese encephalitis, and can be used as an expression vector for thevarious foreign genes.

Those skilled in the art will appreciate that the conceptions andspecific embodiments disclosed in the foregoing description may bereadily utilized as a basis for modifying or designing other embodimentsfor carrying out the same purposes of the present invention. Thoseskilled in the art will also appreciate that such equivalent embodimentsdo not depart from the spirit and scope of the invention as set forth inthe appended claims.

1. A full-length infectious and genetically stable cDNA clone ofJapanese encephalitis virus (JEV), wherein a full-length cDNA of JEV iscloned into a bacterial artificial chromosome (BAC) and an infectiousRNA transcript of JEV is transcribed directly from the cDNA clone;wherein the cDNA clone contains a promoter at the beginning of 5′ end ofa DNA sequence corresponding to a JEV genomic RNA and a restrictionendonuclease recognition sequence at the end of 3′ end of the DNAsequence as a runoff site; and wherein the restriction endonucleaserecognition sequence is XHo I or Xba I.
 2. The cDNA clone as set forthin claim 1, wherein the promoter is SP6 or T7.
 3. The cDNA clone as setforth in claim 1, wherein the restriction endonuclease recognitionsequence does not exist in the JEV genomic RNA.
 4. The cDNA clone as setforth in claim 1, wherein the cDNA clone has a sequence represented bySEQ. ID. No 45, having SP6 promoter or SEQ. ID. No 48 having T7promoter.
 5. The cDNA clone as set forth in claim 1, wherein the cDNAclone is pBAC^(SP6)/JVFLx/XbaI containing the JEV cDNA represented bySEQ. ID. No 45 or pBAC^(T7)/JVFLx/XbaI containing the JEV cDNArepresented by SEQ. ID. No
 48. 6. The cDNA clone as set forth in claim5, wherein the vector is pBAC^(T7)/JVFLx/XbaI having T7 promoter anddeposited under Accession No: KCTC 10346BP.
 7. The cDNA clone as setforth in claim 5, wherein the cDNA clone is pBAC^(SP6)/JVFLx/XbaI havingSP6 promoter and deposited under Accession No: KCTC 10347BP.
 8. The cDNAclone as set forth in claim 1, wherein the JEV genomic RNA consists of a5′ nontranslated region (NTR), a single polypeptide coding region, and a3′ NTR.
 9. A full-length infectious and genetically stable cDNA clone ofJapanese encephalitis virus (JEV), comprising: SEQ. ID. No 45 having SP6promoter, wherein the cDNA clone contains a promoter at the beginning of5′ end of a DNA sequence corresponding to a JEV genomic RNA and arestriction endonuclease recognition sequence at the end of 3′ end ofthe DNA sequence as a runoff site.
 10. A vector, comprising: afull-length infectious and genetically stable cDNA clone of Japaneseencephalitis virus (JEV), wherein the vector is pBAC^(SP6)/JVFLx/XbaI.11. The vector according to claim 10, wherein the vector ispBAC^(SP6)/JVFLx/XbaI having SP6 promoter and deposited under AccessionNo: KCTC 10347BP.
 12. The vector according to claim 10, wherein the JEVcomprises SEQ. ID. No
 45. 13. A full-length infectious and geneticallystable cDNA clone of Japanese encephalitis virus (JEV), comprising: SEQ.ID. No 48 having T7 promoter, wherein the cDNA clone contains a promoterat the beginning of 5′ end of a DNA sequence corresponding to a JEVgenomic RNA and a restriction endonuclease recognition sequence at theend of 3′ end of the DNA sequence as a runoff site.
 14. A vector,comprising: a full-length infectious and genetically stable cDNA cloneof Japanese encephalitis virus (JEV), wherein the vector ispBAC^(T7)/JVFLx/XbaI.
 15. The vector according to claim 14, wherein thevector is pBAC^(T7)/JVFLx/XbaI having T7 promoter and deposited underAccession No: KCTC 10346BP.
 16. The vector according to claim 14,wherein the JEV comprises SEQ. ID. No 48.